Compositions and methods for treating and preventing tissue injury and disease

ABSTRACT

The present invention provides novel compositions comprising multipotent cells or microvascular tissue, wherein the cells or tissue has been sterilized and/or treated to inactivated viruses, and related methods of using these compositions to treat or prevent tissue injury or disease in an allogeneic subject.

RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.15/633,311 filed Jun. 26, 2017; which application is a continuation ofU.S. application Ser. No. 14/429,511 filed Mar. 19, 2015; whichapplication is a National Stage Entry of PCT/US2013/060181 filed Sep.17, 2013; which application claims the benefit of U.S. ProvisionalApplication No. 61/703,203 filed Sep. 19, 2012, the disclosures of whichare expressly incorporated by reference herein.

BACKGROUND Field

Several embodiments of the present invention are directed to novelcompositions comprising multipotent cells and/or microvascular tissue,which has been sterilized and/or treated to inactivate any viruses, andmethods for their preparation and allogeneic or xenogeneic use intreating or preventing tissue injury and diseases, such as, e.g.,arthritis.

Description of the Related Art

Injuries to soft tissues, such as muscles, tendons, ligaments, and jointcapsules, occur quite frequently. Such injuries typically result intissue dysfunction characterized by pain, inflammation and internaltissue stress, and can ultimately result in a functional disability. Forexample, while sprains to tendons will heal spontaneously, completetears of a tendon will often lead to disability if not surgicallytreated. Even despite surgical repair, about 15% of Achilles tendon and40% of two tendon rotator cuff repairs subsequently fail. Furthermore,the repaired tendon seldom returns to pre-injury strength and functionlevels.

Tissue repair generally includes several phases, including an initialinflammatory response followed by cellular proliferation and tissueremodeling. Fundamental processes of tissue repair include bothfibroplasia and angiogenesis. Fibroblasts activated by inflammatorymediators migrate into the wound, proliferate, and lay downcollagen-rich extracellular matrix, while capillaries in the damagedtissue grow towards the repair zone to reestablish blood flow. Duringthe remodeling process, scar tissue is reabsorbed and replaced withdenser, oriented collagen, to produce tissue with some of thecharacteristics of the original tissue.

SUMMARY

A variety of different therapeutic methods to aid in tissue repair havebeen developed. These include physical structures, such as bettersutures, bone anchors, and patches or implants to provide scaffoldingfor tissue ingrowth. In addition, a variety of growth factors have beenused to improve tissue growth and migration to the wound site, as wellas to promote angiogenesis. For example, there are reports of improvedtendon healing using growth factors such as BMP-2, BMP-12, PDGF-BB, andbFGF in preclinical models.

More recently, efforts have been made to use stem cells to promote woundhealing and tissue regeneration. Stem cells are believed to mediatewould healing by any of a variety of different mechanisms, including:modulating the inflammatory process; migrating to damaged tissue andrecruiting other cells, such as endothelial progenitor cells, necessaryfor tissue growth; stimulating the proliferation of repair cells;supporting tissue remodeling over scar formation; inhibiting apoptosis;and differentiating into bone, cartilage, tendon, or ligament tissue.There have been a number of reports describing the use of stem cells forthe treatment or generation of many different tissues. Much of this workhas centered on the use of adipose-derived stem cells and othermultipotent cells, because they are easily obtained in large numbers.However, due to concerns that transplanted allogeneic cells or tissuemay invoke an immune response and ultimately rejection, or transferharmful viruses or other pathogens, this work has focused on the use ofautologous cells. Unfortunately, however, the use of autologous stemcells is inconvenient. It requires two distinct surgical procedures withassociated pain, cost and morbidity, and there are also risks associatedwith shipping the tissue to a laboratory for processing and delays intreatment of the injured patient.

Clearly, there is a need in the art for new therapeutic compositions ofallogeneic stem cells and other multipotent cells useful for thetreatment and repair of tissue injury without causing an undesiredimmune response. The present invention satisfies this need and providesother advantages.

Therefore, there are provided, in several embodiments novelcompositions, methods, kits, and cell populations that are useful, suchas in the repair and/or regeneration of tissue.

In several embodiments, there are provided compositions comprisingisolated multipotent cells or processed microvascular tissue, or a cellmembrane obtained from or derived from said cells or tissue, whereinsaid composition has angiogenic or anti-inflammatory activity, andwherein said composition is sterilized and/or viruses within saidcomposition are inactivated. In particular embodiments, the cells orcomposition have not been cultured. In particular embodiments, less thanor equal to 50% or less than or equal to 10% of the cells present in thecomposition are viable. In particular embodiments, substantially none ofthe cells present in said composition are viable. In certainembodiments, at least 1% of said cells exclude trypan blue. Inparticular embodiments, the composition is dried, lyophilized orcryopreserved. In related embodiments, the composition, including thesterilized, dried, lyophilized, or cryopreserved composition, retainsmeasurable angiogenic or anti-inflammatory activity when stored atapproximately room temperature for at least one month. In certainembodiments, the composition comprises an excipient.

In several embodiments, the compositions disclosed herein furthercomprise an implantable scaffold or matrix, which may be, e.g., abone-derived implant, a biofiber scaffold, a porous resorbable polymer,a hydrogel, a putty comprising tissue product, or a suture. Inparticular, cells, tissue or cell membrane are present on a bone,tendon, or dermal facing surface of said implantable scaffold or matrix.

In certain embodiments, a composition of the present invention isformulated for intravenous administration. However, other routes ofadministration can be used in additional embodiments, including directadministration (either to a tissue surface or by direct injection),intraarterial administration, systemic administration and the like.

In several embodiments, the cells or tissue were obtained from amammalian donor, optionally a human. In one embodiment, the donor was ahealthy mammal at the time the cells or tissue were obtained. In certainembodiments, the cells comprise stem cells or progenitor cells.

In several embodiments, there are provided methods of preparing acomposition comprising isolated multipotent cells, wherein saidcomposition has angiogenic or anti-inflammatory activity, said methodcomprising: dissociating a tissue sample obtained from a donor mammal torelease a plurality of multipotent cells therein; separating a pluralityof the released multipotent cells from one or more other tissuecomponents to produce a composition comprising isolated multipotentcells; optionally drying, lyophilizing, or cryopreserving thecomposition before or after sterilization; sterilizing the compositionand/or inactivating virus present in the composition, before or afterthe optional drying, lyophilizing, or cryopreserving of the composition,wherein the sterilized composition retains measurable angiogenic oranti-inflammatory activity. In several embodiments, the cells orcomposition are not cultured. In certain embodiments, the methodoptionally further comprises filtering the released cells orcomposition, e.g., which may be prior to sterilization or drying,lyophilizing, or cryopreserving. In certain embodiments, thedissociation comprises contacting the tissue sample with one or moreproteases. In particular embodiments, the one or more proteases does notcomprise collagenase. In certain embodiments, the one or more proteasescomprises or consists of: collagenase type 1 and either dispase orthermolysin; or MMP2, MMP 14 and either dispase or thermolysin.Combinations of these proteases (or other functional equivalents) can beused. In additional embodiments, the dissociating or separatingcomprises ultrasonic agitation, filtration, or use of a densitygradient. In one embodiment, the tissue is adipose-derived tissue, andthe ultrasonic agitation, filtration or use of a density gradientseparates said released multipotent cells from adipocytes.

In additional embodiments, there are provided methods of preparing acomposition comprising processed microvascular tissue, wherein saidcomposition has angiogenic or anti-inflammatory activity, said methodcomprising: dissociating a microvascular tissue sample obtained from adonor mammal to produce a composition comprising dissociatedmicrovascular tissue; removing one or more tissue components from thecomposition comprising dissociated microvascular tissue; optionallydrying, lyophilizing, or cryopreserving the composition before or aftersterilization; and sterilizing the composition and/or inactivating viruspresent in the composition before or after the optional drying,lyophilizing, or cryopreserving, wherein the sterilized compositionretains measurable angiogenic or anti-inflammatory activity. Inparticular embodiments, the cells or composition are not cultured. Inparticular embodiments, the method further comprises filtering thecomposition. In particular embodiments, the dissociation comprisescontacting the tissue sample with one or more proteases. In certainembodiments, the one or more proteases does not comprise collagenase. Incertain embodiments, the one or more proteases comprises or consists of:collagenase type 1 and either dispase or thermolysin; or MMP2, MMP 14and either dispase or thermolysin. In certain embodiments, thedissociation or removing comprises ultrasonic agitation, filtration, oruse of a density gradient. In particular embodiments, the microvasculartissue is adipose-derived tissue, and said ultrasonic agitation,filtration or use of a density gradient removes adipocytes from thecomposition.

In an additional embodiment, the present invention provides a moistureimpermeable container comprising a sterile, dry composition, whereinsaid composition comprises isolated multipotent cells or processedmicrovascular tissue, or a cell membrane comprising said cells or tissueor obtained from said cells or tissue, said composition has angiogenicor anti-inflammatory activity, said composition is sterilized and/orviruses within said composition are inactivated, and said compositionretains measurable angiogenic or anti-inflammatory activity when storedat approximately room temperature for at least one month. In certainembodiments, the cells or composition have not been cultured. Inparticular embodiments, less than or equal to 50% or less than or equalto 10% of the cells present in said composition are viable. In certainembodiments, substantially none of the cells present in said compositionare viable. In particular embodiments, at least 1% of said cells excludetrypan blue. In particular embodiments, the composition comprises anexcipient.

In particular embodiments of the moisture impermeable container, thecomposition further comprises an implantable scaffold or matrix. Inparticular embodiments, the implantable scaffold or matrix is abone-derived implant, a biofiber scaffold, a porous resorbable polymer,a hydrogel, a putty comprising tissue product, or a suture. In certainembodiments, the cells, tissue or cell membrane are present on a bone,tendon or dermal facing surface of said implantable scaffold or matrix.

In one embodiment of the moisture impermeable container, the compositionis formulated for intravenous administration.

In particular embodiments of the moisture impermeable container of theinvention, the cells or tissue were obtained from a mammalian donor,optionally a human. In particular embodiments, the donor was a healthymammal at the time the cells or tissue were obtained. In certainembodiments, the cells comprise stem cells or progenitor cells. Incertain embodiments, the container is a vial comprising a hermetic seal.In various embodiments, the container is present within a sealed packagecomprising a sterile interior.

In another related embodiment, the present invention provides a methodof treating or preventing an injury or disease, or promoting tissueregeneration, in a mammal, comprising providing to said mammal acomposition of the present invention or a composition prepared accordingto a method of the present invention. In particular embodiments, thecomposition is surgically implanted into the mammal. In certainembodiments, the composition is implanted within or adjacent to a siteof injury or disease in said mammal. In related embodiments, thecomposition is provided to said mammal intravenously. In certainembodiments, the injury is present in a soft tissue. In particularembodiments, the injury is present in a tendon, a ligament, skin, abone, cartilage, a disc, or microvascular tissue. In particularembodiments, the injury or disease is an ischemic injury, a reperfusioninjury, a microvascular injury, or inflammation. In certain embodiments,the disease is arthritis, such as e.g., osteoarthritis or rheumatoidarthritis.

In additional embodiments, there are provided compositions comprisingmultipotent cells and one or more vessel wall and/or extracellularmatrix components. In another embodiment, the present invention includesa sterilized composition comprising multipotent cells. In furtherembodiments, the present invention includes a composition comprisingmultipotent cells that exclude trypan blue but will not proliferate. Ina further embodiment, the present invention includes a compositioncomprising multipotent cells and one or more vessel wall extracellularmatrix components. In another embodiment, the present invention includesa sterilized composition comprising multipotent cells. In a furtherembodiment, the present invention includes a composition comprisingmultipotent cells that exclude trypan blue but will not proliferate. Ina related embodiment, the present invention includes a compositioncomprising sterilized multipotent cells that exclude trypan blue butwill not proliferate. In certain embodiments of compositions of thepresent invention, at least 50% or at least 90% of the cells present inthe composition exclude trypan blue but will not proliferate. In certainembodiments, the composition comprises multipotent cells. In oneembodiment, at least 50% or at least 90% of the multipotent cellspresent in the composition exclude trypan blue but will not proliferate.In particular embodiments, less than or equal to 50% or less than orequal to 10% of the total cells present in the composition are viable.In particular embodiments, substantially none of the cells present insaid composition are viable. In certain embodiments of any of thecompositions or methods of the present invention, at least 1% or atleast 5%, or at least 10%, or at least 20%, at least 50%, or at least90% of said cells exclude trypan blue.

In an additional embodiment, the present invention includes a sterilizedcomposition comprising two or more components of multipotent cells. Incertain embodiments, the composition comprises five or more or ten ormore components of multipotent cells. In another related embodiments,the present invention includes a composition comprising cell membraneand proteins from multipotent cells. In particular embodiments, thecomposition does not comprise any viable cells or does not comprise anyintact cells.

In addition, the present invention provides that any of the compositionsof the invention may be used in treating or preventing an injury ordisease in a subject, including any of the injuries or conditionsdescribed herein, wherein said multipotent cells are not autologous tosaid subject.

The methods summarized above and set forth in further detail belowdescribe certain actions taken by a practitioner; however, it should beunderstood that they can also include the instruction of those actionsby another party. Thus, actions such as “administering microvasculartissue” include “instructing the administration of microvasculartissue.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic diagram of the studies described in Example1.

FIG. 2 is a graph showing the estimated cell counts per 100×magnification of cells in 6 fields. BMA and BMB cells in Buffers 1&2after and before irradiation (Control) were used to attract HUVEC cellsthat were CM-DiI labeled. The cell count was estimated by a visual countof 6 images (fields) at 100× magnification. The counts were averaged andplotted a shown. The dotted lines represent the average for the negativemedia controls. Cell numbers above that line represent increasedmigration due to the BMA or BMB material samples. The labels shown topto bottom correspond to the bars shown left to right for each timepoint.

FIG. 3 shows fluorescence microscopy images of labeled human endothelialcells in the presence of BMA in Buffer 1 over a 12, 24 and 48 hour timecourse of transmigration.

FIG. 4 shows fluorescence microscopy images of labeled human endothelialcells in the presence of BMB in Buffer 1 over a 12, 24 and 48 hour timecourse of transmigration.

FIG. 5 shows fluorescence microscopy images of labeled human endothelialcells in the presence of BMA in Buffer 2 over a 12, 24 and 48 hour timecourse of transmigration.

FIG. 6 shows fluorescence microscopy images of labeled human endothelialcells in the presence of BMB in Buffer 2 over a 12, 24 and 48 hour timecourse of transmigration.

FIG. 7 shows fluorescence microscopy images of labeled human endothelialcells. The sample on the left is undiluted EZ-CPZ™, which isrepresentative of buffers 1 & 2 in which the cells were preserved.EZ-CPZ™ is a cryopreservation media (Incell Corp., San Antonio, Tex.).EZ-CPZ™ is described by Incell Corp. as a ready-to-use, serum-free, andprotein-free cryopreservation medium that is gently mixed 1:1 (v:v) witha cell suspension. EZ-CPZ™ supports high viability and re-animation of avariety of cell types including: primary cell cultures, lymphocytes,hybridoma, CHO, colon, IBHK and cancer cell lines. EZ-CPZ™ contains aproprietary formulation of clinical grade components, vitrification andcryopreservation agents, and a final concentration of 5% DMSO. EZ-CPZ™provides cryoprotection to human and other mammalian cells. On the rightis a 50:50 mixture of EZ-CPZ and M3D™ defined media (Incell Corp., SanAntonio, Tex.), which is representative of the buffers in which theprocessed microvascular tissue composition material will be preservedin. M3D™ is described by Incell Corp. as a defined medium that containssalts, amino acids, and sugars, but no growth factors or undefinedcomponents such as serum or extracts. The time course of transmigrationwas imaged at the 12 and 48 hour time points.

FIG. 8 shows fluorescence microscopy images. SVF cells were plated in a48 well plate and grown to approximately 50% confluence. BMA materialwas stained with CMDiI and rinsed. 50 μl of the BMA material was putonto a single well of growing SVF cells. Fluorescence is observed wherethe SVF cells have taken up the CMDiI stained material and incorporatedit into their own membranes. BMA material in Buffer 1 is depicted in thetop row, and BMA material in Buffer 2 is depicted in the bottom row. Thematerial on the left side was irradiated and the material on the rightside (Cont) was not irradiated.

FIG. 9 shows fluorescence microscopy images. SVF cells were plated in a48 well plate and grown to approximately 50% confluence. BMB materialwas stained with CMDiI and rinsed. 50 μl of the BMA material was putonto a single well of growing SVF cells. Fluorescence is observed wherethe SVF cells have taken up the CMDiI stained material and incorporatedit into their own membranes. BMB material in Buffer 1 is depicted in thetop row, and BMB material in Buffer 2 is depicted in the bottom row. Thematerial on the left side was irradiated and the material on the rightside (Cont) was not irradiated.

FIG. 10 depicts data related to the migration of human umbilical veinendothelial cells (HUVEC) cells in response to exposure to microvasculartissue. The number of HUVEC's crossing the membrane of a Transwell platewas counted at 48 hrs and compared to culture media+EGF controls.

FIG. 11 depicts data related to the restoration of blood flow to thehindlimbs of mice after femoral artery transection at day 0, and days 7and 14 after administration of either lyophilized or sterilizedmicrovascular tissue.

FIG. 12 depicts data related to the generation of new blood vessels inSCID mice after injections with matrigel alone, or in combination witheither lyophilized or sterilized microvascular tissue.

FIG. 13A-13C relate to the regeneration of bone after implantation ofmicrovascular tissue. FIG. 13A depicts data related to the strength,elastic modulus, and toughness (as compared to control contralateralbone) after administration of a scaffold with or without microvasculartissue. FIG. 13B deptics histologic data regarding bone regenerationafter administration of scaffold alone. FIG. 13C deptics histologic dataregarding bone regeneration after administration of scaffold incombination with microvascular tissue.

FIG. 14A-14H relate to the regeneration of cartilage after implantationof microvascular tissue. FIG. 14A shows a macroscopic image of cartilagetreated with scaffold alone, while FIGS. 14B, 14C, and 14D show imagesrelated to the fill in of the induced cartilage defects, theproteoglycan retention and matrix staining (respectively) in cartilagetreated with scaffold alone. FIG. 14E shows a macroscopic image ofcartilage treated with scaffold supplemented with microvascular tissue,while FIGS. 14F, 14G, and 14H show images related to the fill in of theinduced cartilage defects, the proteoglycan retention and matrixstaining (respectively) in cartilage treated with scaffold supplementedwith microvascular tissue.

FIG. 15A-15F related to repair of abraded tendon using microvasculartissue. FIGS. 15A and 15B depict, respectively, Masson's Trichromestaining or Tenascin immunohistochemistry of abraded and untreatedtendon. FIGS. 15C and 15D depict, respectively, Masson's Trichromestaining or Tenascin immunohistochemistry of abraded tendon treated withscaffold alone. FIGS. 15E and 15F depict, respectively, Masson'sTrichrome staining or Tenascin immunohistochemistry of abraded tendontreated with scaffold supplemented with microvascular tissue.

FIG. 16 shows data for Patient A, who presented with a diabetic footulcer on the bottom of his foot that had not healed despite standardcare with hydrocolloid dressings. After debridement, the ulcer measured5.1 cm² and the neuropathy extended halfway to the knee. Four weeksafter treatment with the therapeutic agent of the instant invention, theDFU measured 3.5 cm² and the extent of the neuropathy was reduced byabout 14 cm.

FIG. 17 shows data for Patient B, who presented with a diabetic footulcer on the medial side of her foot that had not healed despitestandard care with hydrocolloid dressings. After debridement the ulcermeasured 6.1 cm² and the neuropathy extended over halfway to the knee.Four weeks after treatment with the therapeutic agent of the instantinvention, the DFU measured 0.4 cm² and the extent of the neuropathy wasreduced by about 16 cm.

FIG. 18 shows control data. Patient C presented with a diabetic footulcer on the bottom of her foot that had not healed despite standardcare with hydrocolloid dressings. After debridement the ulcer measured4.0 cm² and the neuropathy extended ⅓ of the distance to the knee. Fourweeks after treatment as with Patient A and B but without the additionof multipotent cells, the DFU measured 1.6 cm² and the extent of theneuropathy was reduced by about 1 cm.

DETAILED DESCRIPTION

The present invention is based, in part, on the development of novelmethods for processing microvascular tissue to produce a compositioncomprising isolated multipotent cells or processed microvascular tissue,or a cell membrane comprised of said cells or tissue. In variousembodiments, the cells or tissue are not cultured during theseprocedures. Advantageously, the composition has angiogenic oranti-inflammatory activity. In several embodiments, the composition issterilized and/or viruses within said composition are inactivated duringthe procedures, yet the composition still displays unexpectedtherapeutic efficacy.

The novel compositions produced by the methods of the present inventionoffer advantages over prior processed microvascular tissue andmultipotent cell compositions, including advantages associated withtreating or preventing an injury, e.g., a soft tissue injury, in asubject. These advantages include (but are not limited to): (1) theability to use the compositions of the present invention for theallogeneic or xenogeneic treatment of subjects; (2) compositions of thepresent invention produce a reduced immune response and reducedlikelihood of rejection; (3) compositions of the present invention haveanti-inflammatory activity; (4) compositions of the present inventionhave angiogenic activity; (5) compositions of the present invention aresterile and/or are not contaminated by harmful viruses; and (6)compositions of the present invention may be stably stored prior to useand/or are ready for immediate use. In short, these compositionsconveniently provide all the mechanisms of action inherent intraditional, viable stem or multipotent cell preparations except fordifferentiation into tissues. In the following description, certainspecific details are set forth in order to provide a thoroughunderstanding of various embodiments of the invention. However, oneskilled in the art will understand that the invention may be practicedwithout these details.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. For the purposes of thepresent invention, the following terms are defined below.

The words “a” and “an” denote one or more, unless specifically noted.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length. In any embodiment discussed in the context ofa numerical value used in conjunction with the term “about,” it isspecifically contemplated that the term about can be omitted.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

By “consisting essentially of” is meant including any elements listedafter the phrase, and limited to other elements that do not interferewith or contribute to the activity or action specified in the disclosurefor the listed elements. Thus, the phrase “consisting essentially of”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

As used herein, the terms “function” and “functional”, and the like,refer to a biological, enzymatic, or therapeutic function.

An “increased” or “enhanced” amount is typically a “statisticallysignificant” amount, and may include an increase that is 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times)(including all integers and decimal points in between and above 1, e.g.,2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein.

A “decreased” or “reduced” or “lesser” amount is typically a“statistically significant” amount, and may include a decrease that isabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100,500, 1000 times) (including all integers and decimal points in betweenand above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or leveldescribed herein.

By “obtained from” is meant that a sample such as, for example, a cellor tissue, is isolated from, or derived from, a particular source, suchas a desired organism or a specific tissue within a desired organism.

As used herein, the term “isolated”, e.g., with respect to a multipotentcell, means removed from its natural environment. For example, a cell isisolated if it is separated from some or all of the coexisting materialsin its natural environment.

The term “processed microvascular tissue” as used herein refers tomicrovascular tissue that is dissociated as described herein.

The term “cryopreserved” as used herein refers to multipotent cell orprocessed microvascular tissue compositions that are frozen, e.g., atlow temperature. Processed microvascular tissue and cryopreservedmultipotent cell and microvascular tissue compositions have a variety ofbiological properties, including anti-inflammatory activity andangiogenic activity.

“Multipotent cells” refers to cells that maintain the capacity todifferentiate into two or more different specialized cell types.“Multipotent cells” include stem cells and multipotent progenitor cells.As used herein, the term “multipotent cell” refers to a cell's originalcapacity to differentiate into two or more different specialized celltypes prior to it being sterilized or preserved according to a methoddescribed herein. Examples of multipotent cells include, but are notlimited to, mesenchymal stem cells, embryonic stem cells, neural stemcells, endothelial progenitor cells, adipose-derived stem cells, andumbilical cord stem cells. It is understood that following sterilizationor preservation according to the methods described herein, a multipotentcell may lose its capacity to grow or differentiate.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

A “pharmaceutical composition” refers to a formulation of a compositionof the invention and a medium generally accepted in the art for thedelivery of a therapeutic agent to mammals, e.g., humans. Such a mediumincludes any pharmaceutically acceptable carriers, diluents orexcipients therefore.

As used herein, unless the context makes clear otherwise, “treatment,”and similar words such as “treated,” “treating” etc., indicates anapproach for obtaining beneficial or desired results, including clinicalresults. Treatment can involve optionally either the reduction oramelioration of symptoms of an injury, disease or condition, or thedelaying of the progression of the injury, disease or condition.Administration of a composition described herein may, in someembodiments, treat one or more of such symptoms.

As used herein, unless the context makes clear otherwise, “prevention,”and similar words such as “prevented,” “preventing” etc., indicates anapproach for preventing, inhibiting or reducing the likelihood of theonset or recurrence of an injury, disease or condition. It also refersto preventing, inhibiting or reducing the likelihood of the occurrenceor recurrence of one or more symptoms of an injury, disease orcondition, or optionally an approach for delaying the onset orrecurrence of an injury, disease or condition or delaying the occurrenceor recurrence of one or more symptoms of an injury disease or condition.As used herein, “prevention” and similar words also includes reducingthe intensity, effect, symptoms and/or burden of an injury, disease orcondition.

As used herein, an “effective amount” or a “therapeutically effectiveamount” of a composition is that amount sufficient to affect a desiredbiological effect, such as, e.g., beneficial clinical results.

The terms “autologous transfer,” “autologous transplantation,” and thelike refer to treatments wherein the tissue donor is also the recipientof the composition produced from the tissue.

The terms “allogeneic transfer,” “allogeneic transplantation,” and thelike refer to treatments wherein the tissue donor is of the same speciesas the recipient of the composition produced from the tissue, but is notthe same individual.

The terms “xenogeneic transfer,” “xenogeneic transplantation,” and thelike refer to treatments wherein the tissue donor is of a differentspecies than the recipient of the composition produced from the tissue.

Methods of Producing Stem Cell and Microvascular Tissue Compositions

Aspects of the present invention relate to novel methods of processingvascular, e.g., microvascular, tissue to produce a compositioncomprising multipotent cells or fragments thereof. In particularembodiments, the composition further comprise one or more additionaltissue components. Accordingly, the term “processed microvascular tissuecomposition” refers to compositions of the present invention, which mayor may not comprise intact multipotent cells. In particular embodiments,a microvascular tissue composition of the present invention does notcomprise any intact multipotent cells or does not comprise any livemultipotent cells or does not comprise any live cells. In certainembodiments, a microvascular tissue composition of the present inventioncomprises fragments or cell membranes of multipotent cells. A“composition comprising multipotent cells” or “multipotent cellcomposition” of the present invention may comprise live and/or deadmultipotent cells.

Several embodiments provide novel methods for producing multipotent celland microvascular tissue compositions, including those useful in thetreatment and prevention of various injuries, disease or pathologicalconditions, e.g., a soft tissue injury. In particular embodiments, themethods of the present invention include sterilizing the isolatedmultipotent cells or microvascular tissue composition and/orinactivating viruses within said cells or tissue. It is a surprising andunexpected finding that such sterilized multipotent cell andmicrovascular tissue compositions retain desirable biologicalproperties, including properties useful in treating or preventinginjury, disease and other pathological conditions.

The multipotent cell and microvascular tissue compositions of thepresent invention may be prepared from any mammalian tissue, e.g.,tissue obtained from a mammal, such as a human, a non-human primate, adog, a cat, or a horse. The compositions of the present invention may beused to treat an autologous, allogeneic or xenogeneic subject.Accordingly, tissue may be obtained from the subject to be treated, orfrom a different donor animal, which may be the same or a differentspecies as the subject to be treated. In particular embodiments, thetissue is obtained from an allogeneic donor of the same species as thesubject to be treated, e.g., a human or non-human mammalian donor. Inparticular embodiments, a donor animal is a healthy donor.

In various embodiments, multipotent cell or microvascular tissuecompositions are prepared from any of a number of different tissues. Inparticular embodiments, the tissue is non-embryonic tissue. For example,in particular embodiments, the tissue used to prepare the compositionsof the present invention is a vascular tissue or a microvascular tissue,such as, e.g., adipose tissue, skin, bone, tendon tissue, post-partumtissue (e.g., umbilical cord tissue or placental tissue), bone marrow,or muscle tissue.

In certain embodiments, compositions of the present invention may beprepared by a method comprising: dissociating a tissue sample to releasecells and/or other tissue components; separating at least a portion ofthe released cells and/or tissue components from one or more othertissue components; and sterilizing the separated cells and/or tissuecomponents and/or treating the separated cells and/or tissue componentsto inactivate viruses therein. In certain embodiments, the separatedcells and/or tissue components are dried, lyophilized, frozen, orcryopreserved before, during or after being sterilized or treated toinactivate viruses. In particular embodiments, the separated cellsand/or tissue components are sterilized or treated to inactivate virusesafter being dried, lyophilized, frozen, or cryopreserved. In relatedembodiments, the separated cells and/or tissue components are sterilizedor treated to inactivate viruses after being contacted with acryoprotectant, e.g., a cryoprotectant that protects cells fromsterilizing radiation. Cryoprotectants can protect cell components bystabilizing proteins, quenching free radicals, and resisting oxidation.Damage can be further minimized by cooling the composition duringradiation, removing oxygen from the composition (e.g., drying thecomposition and/or irradiating in a vacuum or inert atmosphere).

In related embodiments, methods of the present invention comprise:dissociating a tissue sample obtained from a donor mammal to release aplurality of multipotent cells therein; separating a plurality of thereleased multipotent cells from one or more other tissue components toproduce a composition comprising isolated multipotent cells; andsterilizing the composition comprising isolated multipotent cells and/orinactivating virus present in the composition comprising isolatedmultipotent cells. In particular embodiments, the composition comprisingmultipotent cells is dried, lyophilized, frozen, or cryopreservedbefore, during or after being sterilized or treated to inactivateviruses. In particular embodiments, the separated cells and/or tissuecomponents are sterilized or treated to inactivate viruses after beingdried, lyophilized, frozen, or cryopreserved. In related embodiments,the separated cells and/or tissue components are sterilized or treatedto inactivate viruses after being contacted with a cryoprotectant, e.g.,a cryoprotectant that protects cells from sterilizing radiation.

In further related embodiments, methods of the present inventioncomprise: dissociating a tissue sample obtained from a donor mammal torelease a plurality of tissue components therein; separating a pluralityof released tissue components to produce a composition comprising one ormore tissue components; and sterilizing the composition and/orinactivating virus present in the composition. In particularembodiments, the composition is dried, lyophilized, frozen, orcryopreserved before, during or after being sterilized or treated toinactivate viruses. In particular embodiments, the separated cellsand/or tissue components are sterilized or treated to inactivate virusesafter being dried, lyophilized, frozen, or cryopreserved. In relatedembodiments, the separated cells and/or tissue components are sterilizedor treated to inactivate viruses after being contacted with acryoprotectant, e.g., a cryoprotectant that protects cells fromsterilizing radiation.

In certain embodiments, tissue components comprise one or moremultipotent cells, differentiated cells, components of the extracellularmatrix, growth factors, angiogenic agents, anti-inflammatory agents,cytokines, chemokines, and/or differentiation agents. Extracellularmatrix components include but are not limited to extracellular matrixproteins, such as various collagens, fibronectin, vitronectin, andthrombospondin, and others described herein.

Tissue samples may be obtained from a subject or donor by a variety ofdifferent methods, including surgery, lipoaspiration, biopsy or needlebiopsy. A donor may be alive or dead, e.g., recently deceased.

Tissue may be dissociated by various methods, including both mechanicaland/or enzymatic processing. For example, tissue may be dissociated bymechanical force (mincing or shear forces), enzymatic digestion withsingle or combinatorial proteolytic enzymes, such as a matrixmetalloprotease and/or neutral protease, for example, collagenase,trypsin, dispase, LIBERASE (Boehringer Mannheim Corp., Indianapolis,Ind.), hyaluronidase, and/or pepsin, or a combination of mechanical andenzymatic methods. In particular embodiments, methods of the presentinvention do not employ the use of a collagenase.

In certain embodiments, enzymatic digestion methods employ a combinationof enzymes, such as a combination of a matrix metalloprotease and aneutral protease. In particular embodiments, the matrix metalloproteasemay be a collagenase, and the neutral protease may be thermolysin ordispase. Collagenase may be type 1, 2, 3, or 4 (MMP 1, 8, 13, 18). Inparticular embodiments, enzymatic digestion methods employ a combinationof a matrix metalloprotease, a neutral protease, and a mucolytic enzymefor digestion of hyaluronic acid, such as a combination of collagenase,dispase, and hyaluronidase or a combination of LIBERASE (BoehringerMannheim Corp., Indianapolis, Ind.) and hyaluronidase. Other enzymesknown in the art for cell dissociation include papain,deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin,gelatinases, or elastase, that may be used either on their own or incombination with other enzymes such as matrix metalloproteases,mucolytic enzymes, and neutral proteases. In certain embodiments, acombination of enzymes comprises or consists of Type 1 collagenase witheither dispase or thermolysin, Liberase, and/or Vitacyte. In certainembodiments, a combination of enzymes comprises or consists of Type 1collagenase with either dispase or thermolysin, Liberase, and/or Cizyme.In particular embodiments, a collagenase is not used, either alone or incombination with one or more additional enzymes. In certain embodiments,MMP 2 and/or 14 are used instead of MMP 1 (alone or in any of thecombinations described herein.

The temperature and period of time tissues or cells are in contact withproteases to achieve dissociation is known and may be readily determinedby one of skill in the art. The enzymatic digestion process can beadjusted to increase or decrease cell dissociation. For example, if morecomplete cell dissociation is desired, more than one enzyme can beincluded or digestion time can be increased. While cell viability neednot be maintained, in some embodiments it is generally desired thatcellular membranes remain generally intact to preserve membranescontaining attachment and signaling molecules even if some cell lysisoccurs during enzymatic digestion. Thus, the use of enzymes such aslipidases may not be useful in such a process, according to oneembodiment of the present invention.

Alternatively, or in addition to enzymatic treatment, tissue can bedissociated using a non-enzymatic method. For example, tissue can bedissociated using physical or chemical means, including the use ofchelators, ultrasonic agitation, ultrasound (e.g., to lyse or removeadipocytes), or mechanical cell dissociation.

In particular embodiments where the tissue is bone, the bone isdemineralized prior to enzymatic (or other) processing to free cellsfrom the collagen matrix. In particular embodiments, the bone isdemineralized using EDTA (as opposed to a solvent to defat the tissuefollowed by acid to remove bone minerals). In certain embodiments,methods of processing tissue, e.g., bone, do not include one or more ofthe following: solvent extraction of fats and/or cells, cryo-milling toreduce particle size, and/or acid demineralization.

Following tissue dissociation, the dissociated tissue can be furthertreated to isolate or separate desired tissue components, e.g.,multipotent cells (and/or other desired cellular or non-cellular tissuecomponents), from undesired tissue components. These methods may beused, e.g., to remove undesired cells or molecules, such as red bloodcells, adipocytes, other differentiated cells, or lipids. A variety ofmethods may be used to separate mutipotent cells and other desiredtissue components from undesired cells or tissue components, such as,e.g., filtration (e.g., a 20 micron pore size filter would passmultipotent cells but retain many adipocytes or muscle cells),centrifugation (adipocytes and lipids float, while multipotent cells arepelleted), or density gradients (gradients may be used to pellet redcells and to suspend multipotent cells at different levels than unwantedcells). The particular method used may depend, in part, upon the sourceof tissue being processed. For example, if the tissue source is adiposetissue, the dissociated tissue is optionally centrifuged at relativelylow force to separate lipids, adipocytes, and some pre-adipocytes fromother components of the microvascular tissue, while a density gradientis optionally used when isolating multipotent cells from bone marrow. Inother embodiments, muscle cell isolation protocols, such as the use ofdensity gradient centrifugation, may be used to further treat muscletissue following enzymatic digestion to remove muscle cells and enrichfor desired cells.

In certain embodiments, a composition of the present invention is alsofiltered, e.g., to remove clumps. For instance, filtration, e.g., with a20 to 50 micron pore size filter, to remove large clumps may beperformed during preparation of a composition for intravenous injection,to avoid clogging of capillaries. In particular embodiments, filtrationis performed after dissociation and prior to use, e.g., afterdissociation and prior to lyophilization or sterilization.

Depending on the embodiment, the multipotent cell composition andmicrovascular tissue compositions are optionally frozen or dried forstorage or preservation, e.g., dried (e.g., freeze-dried orspray-dried), cryopreserved, or frozen. Any appropriate excipient can beused when preserving compositions of the invention, including sugars(e.g., trehalose, mannitol, sucrose), polyalcohols (e.g., polyethyleneglycol), aldehydes, proteins (e.g., albumin), amino acids (e.g.,glycine), surfactants (e.g., Tween 20), DMSO, and/or permanganates. Inseveral embodiments, no excipient is used.

Cells and their active components can be protected in part from damageby ionizing radiation by several methods. Antioxidants or free radicalscavengers can be very effective. Agents that immobilize macromoleculessuch as the sugars used in lyophilization and the drying process itselfincrease the odds that disrupted chains will recombine in their originalchemical structure. Removal of water, air and other sources of oxygenwill reduce the oxidation of proteins or other biologically activespecies. Freezing the composition during radiation also reduces the oddsof cleaved molecules recombining inappropriately. Finally, the muchgreater concentration of excipients than cells or active molecules inthe cells means that there will be fewer cleavages of active compoundssimply due to mass action effects.

Freeze drying (e.g., lyophilization) typically involves four steps:pretreatment, freezing, primary drying, and secondary drying.Pretreatment can include concentration adjustment or the addition of oneor more excipients. Following pretreatment, the multipotent cell ormicrovascular tissue composition is frozen. The freezing step istypically done in a carefully controlled manner (e.g., at a rate ofcooling of between about −0.5° C. per minute to about −50° C. perminute) to preserve cell structure, however cell viability need not bepreserved. In some embodiments, the multipotent cell or microvasculartissue composition is frozen at a rate of cooling of about −10° C. perminute. The rate of cooling can be adjusted based on the particularcells or tissue and excipients used. The multipotent cell ormicrovascular tissue composition can be frozen using any appropriatemeans, including using mechanical refrigeration and/or exposing acontainer containing the composition to dry ice or liquid nitrogen untilit reaches a temperature suitable for freeze drying. During the primarydrying step, the temperature and pressure are adjusted to provideconditions suitable to cause sublimation of water from the multipotentcells or microvascular tissue. The specific temperature and pressure canbe adjusted to accommodate the excipient used and/or the concentrationof the cells or microvascular tissue. During the secondary drying step,the temperature and pressure can be further adjusted to facilitate theremoval of unfrozen water from the multipotent cells or microvasculartissue. The final water content following the secondary drying step ispreferably between about 1% and about 4% by weight (including about 1%to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% toabout 5%, and overlapping ranges thereof), but can be adjusted in orderto maximize shelf life or biological activity.

In some embodiments, the multipotent cell or microvascular tissuecomposition is spray dried. Prior to spray drying, the multipotent cellor microvascular tissue composition can be pretreated similarly tomultipotent cell or microvascular tissue composition that is to befreeze dried, with the excipients being chosen as appropriate for spraydrying rather than freeze drying. During spray drying, the multipotentcells or microvascular tissue is atomized into droplets and exposed toheated air in a drying chamber. In one embodiment, an excipient is asugar that does not melt under the temperatures utilized. In particularembodiments, an excipient is an antioxidant, such as BHA, BHT, andpropyl gallate, for example.

In some embodiments, multipotent cell and microvascular tissuecompositions are not processed by drying, but instead are cryopreserved.Methods for cryopreserving cells and tissue are known. For example,cells or microvascular tissue compositions may be mixed with one or moreexcipients or cryoprotectants (e.g., DMSO, PEG, albumin, or sugar) andcooled in a carefully controlled manner. In some embodiments, cooling isdone in two or more stages in which the first stage is done in acontrolled manner (e.g., reducing the temperature by 1° C. per minute)to an intermediate temperature (e.g., −30° C.), with the second stagetransferring cells or tissue at the intermediate temperature to a colderstorage temperature (e.g., −196° C.).

Cryopreserved multipotent cell and vascular tissue compositions may bestored at a temperature suitable for maintaining the cryopreserved state(e.g., from about −30° C. to −196° C.). Freeze-dried or spray-driedprocessed cells or microvascular tissue can be stored in a wider varietyof conditions than cryopreserved cells, live cells, or fresh tissue.Suitable temperatures for the storage for processed cells ormicrovascular tissue include temperatures from about −100° C. to about45° C. In some embodiments, freeze-dried or spray-dried processed cellsor microvascular tissue can be stored at room temperature.

In various embodiments, the shelf life of the provided processed cellsor microvascular tissue is at least about one week, at least about onemonth, at least about two months, at least about six months, or greaterwhile maintaining one or more biological activities. In particularembodiments, the composition retains measurable angiogenic oranti-inflammatory activity when stored at approximately 4° C. for atleast one about month, at least about two months, at least about fourmonths, at least about six months, or at least one year. In particularembodiments of kits and compositions described herein, the compositionretains measurable angiogenic or anti-inflammatory activity when storedat approximately −20° C. for at least one about month, at least abouttwo months, at least about four months, at least about six months, or atleast about one year. In particular embodiments, the measurableangiogenic or anti-inflammatory activity is at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, or at leastabout 90% of the activity prior to storage, when measured in an in vivoor in vitro assay, including any of those described herein.

Dissociated tissue, or cells and other tissue components isolatedtherefrom, including the resulting compositions, are optionallysterilized, e.g., to reduce or eliminate contamination bymicroorganisms, such as, e.g., bacterial, viruses, and fungi, or prions.In particular embodiments, compositions comprising multipotent cellsand/or other tissue components, are sterilized using irradiation.Methods of sterilization exist using radiation such as electron beams,X-rays, gamma rays, or ultraviolet radiation. In particular embodiments,sterilization is performed by exposing dissociated tissue, or cells andother tissue components isolated therefrom, to gamma radiation at adosage in the range of about 0.5 to about 5.0 Mrad, or about 1.0 toabout 3.0 Mrad, or about 1.0 Mrad, or about 1.5 Mrad, or about 2.0 Mrad,or about 2.5 Mrad, or about 3.0 Mrad, or about 3.5 Mrad, or about 4.0Mrad, or about 4.5 Mrad, or about 5.0 Mrad (or any amount of gammaradiation between those values). In particular embodiments,sterilization is performed by exposing dissociated tissue, or cells andother tissue components isolated therefrom, to electron beam radiationat a dosage in the range of about 0.5 to about 5.0 Mrad, or about 1.0 toabout 3.0 Mrad, or about 1.0 Mrad, or about 1.5 Mrad, or about 2.0 Mrad,or about 2.5 Mrad, or about 3.0 Mrad, or about 3.5 Mrad, or about 4.0Mrad, or about 4.5 Mrad, or about 5.0 Mrad (or any amount of gammaradiation between those values). It is often easier to measure theamount of radiation to which the compositions are exposed. In particularembodiments, E-beam or gamma radiation levels for sterilization areabout 9 to about 30 kGy, or about 20 to about 30 kGy, or about 9 toabout 17 kGy (or any amount of radiation between those values). Inaddition, dissociated tissue, or cells and other tissue componentsisolated therefrom, may be treated to inactivate viruses. Methods ofinactivating viruses are known in the art, including the use ofirradiation, as described above for sterilization. Other methods ofinactivating viruses may be used, including acid or base treatments,bleach, aldehyde or ethylene oxide solutions, or heat. It is understoodthat cryprotectants and other excipients used for lyophilizing orfreezing the composition may also protect against radiation. Forexample, sugars and albumin (or other stabilizing proteins) along withthe low temperature protect against radiation damage to cells.Accordingly, in particular embodiments, sterilization or viralinactivation is performed after lyophilization.

Additionally, because viability is not required for suitability of theprocessed or cryopreserved multipotent cell or microvascular tissuecompositions for therapeutic use, the preservation process and storageneed not be adjusted to maintain viability. The percentage of viablecells in the provided multipotent cell or microvascular tissuecompositions before processing, sterilization, or cryopreservation canbe up to 100%. After processing, sterilization, or cryopreservation, itmay be less than about 50%, e.g., less than about 40%, less than about30%, less than about 20%, less than about 10%, or less than about 1%. Insome embodiments, the provided processed multipotent cell ormicrovascular tissue composition contains no viable cells afterprocessing, sterilization, or cryopreservation. In several embodiments,the processed or cryopreserved multipotent cell or microvascular tissuecompositions are used in the therapeutic repair and/or regeneration of,for example, soft tissue. In additional embodiments, the processed orcryopreserved multipotent cell or microvascular tissue compositions areused in the therapeutic repair and/or regeneration of hard tissue.

Furthermore, to reduce the likelihood of microbial contamination, donorscan be screened for a predetermined list of microbial organisms (e.g.,HIV, HPV, EBV, TB, etc.) prior to tissue procurement or processing.Screening can be done using known techniques, such as detecting thepresence of a microbial nucleic acid using polymerase chain reaction, orby detecting the presence of a molecule associated with a particularmicrobe by ELISA. Microbially contaminated microvascular tissue can beexcluded from use, according to some embodiments of the presentinvention. In addition, processed or cryopreserved tissue can beproduced using aseptic or sterile techniques.

In particular embodiments, the methods of the present invention do notinclude culturing the dissociated cells or microvascular tissue.

Isolated Stem Cell and Microvascular Tissue Compositions

The methods of the present invention produce unique multipotent cell andmicrovascular tissue compositions. The multipotent cell andmicrovascular tissue compositions provided herein comprise, in severalembodiments, minimally processed, uncultured cells or unculturedmicrovascular tissue (or components thereof) that may include a mixtureof stem and/or progenitor cells produced from the dissociation (e.g., byenzymatic digestion) of a microvascular tissue (e.g., adipose, tendon,or muscle tissue). Processed multipotent cell and microvascular tissuecompositions can include additional molecules (e.g., whole or fragmentedextracellular matrix molecules or growth factors). In addition,processed multipotent cell and microvascular tissue compositions may,depending on the embodiment, comprise fragments or membranes ofmultipotent cells. Furthermore, processed microvascular tissue may ormay not comprise intact multipotent cells.

As noted above, the methods of the present invention may be used toprepare a composition comprising multipotent cells or processedmicrovascular tissue, alone or in combination with one or moreadditional cell types and/or other components.

In particular embodiments, the additional cell type is a stromal,epithelial, or blood-derived cell, including, but not limited to,fibroblasts, keratinocytes including follicular outer root sheath cells,endothelial cells, pericytes, red blood cells, monocytes, lymphocytesincluding plasma cells, neutrophils, thrombocytes, mast cells,adipocytes, muscle cells, hepatocytes, nerve and neuroglia cells,osteocytes, and osteoblasts.

In particular embodiments, the additional tissue component is acomponent of the extracellular matrix. The extracellular matrixcomprises diverse constituents such as glycoproteins, proteoglycans,complex carbohydrates, and other molecules. The extracellular matrix maycomprise any of a number of different proteins, including variouscollagens, elastin, fibronectin, laminin, proteoglycans, vitronectin,thrombospondin, tenascin (cytoactin), entactin (nidogen), osteonectin(SPARC), anchorin CII, chondronectin, link protein, osteocalcin, bonesialoprotein, osteopontin, epinectin, hyaluronectin, amyloid Pcomponent, fibrillin, merosin, s-laminin, undulin, epilligrin, kalinin,fibrin, fibrinogen, and HSP.

In related embodiments, the additional tissue component comprises agrowth factor, an angiogenic agent, an anti-inflammatory agent, acytokine, or a differentiation agent. For example, a growth factor orangiogenic agent may be selected from basic fibroblast growth factor,other fibroblast growth factors, bone morphogenetic proteins, hepatocytegrowth factor, keratinocyte growth factor, granulocyte macrophage colonystimulating factor, platelet-derived growth factor, transforming growthfactor β1 and/or β3, vascular endothelial cell growth factor. Additionalgrowth factors and classes or families of growth factors that may beused include any of those listed in Table 15, which also includesrepresentative biological activities for certain growth factors.

In particular embodiments, compositions of the present invention containno or substantially no adipose tissue, bone mineral, muscle cells,and/or blood cells, e.g., one or more of these cell types or bonemineral was removed from the composition during processing. This canincrease the concentration of cells associated with the microvasculaturein the composition. In particular embodiments, compositions of thepresent invention comprise DNA or a substantial amount of DNA, e.g., DNAwas not removed from the composition during processing, for example, thetissue was not decellularized and nor was DNA washed out. In particularembodiments, a composition of the present invention is a water-solublesuspension of cells and/or tissue components. In certain embodiments, acomposition of the present invention, alone, does not comprise astructural scaffold or matrix, such as, e.g., a dermal or tendon graft.In particular embodiments, a composition of the present invention may beproduced using microvascular tissues such as skin, umbilical cord, orbone, which are treated to free cells from the matrix.

In particular embodiments, a composition of the present invention hasone or more biological activities. For example, in certain embodiments,a composition has anti-inflammatory or angiogenic activity. In certainrelated embodiments, a composition promotes blood vessel formation ortissue healing. Combinations of these effects are achieved in severalembodiments.

In certain embodiments, a composition of the present invention hasanti-inflammatory activity. In particular embodiments, an injured ordiseased tissue (e.g., an injured or diseased tissue undergoing aninflammatory response) exposed to or contacted with a composition of thepresent invention exhibits reduced inflammation as compared to when theinjured or diseased tissue is similarly treated but not exposed to orcontacted with the composition of the present invention. In certainembodiments, the amount of inflammation in the tissue exposed to orcontacted with the composition of the present invention is reduced by atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, as compared to the amount ofinflammation when the injured or diseased tissue is not exposed to orcontacted with the composition of the present invention. Inflammationmay be measured by means available in the art, including, e.g., thenumber of lymphocytes observed in the affected tissue when observedhistologically.

In particular embodiments, a composition of the present invention hasanti-inflammatory activity that may be measured in an in vitro assay. Incertain embodiments, the amount of inflammation measured in an in vitroassay in the presence of a composition of the present invention is atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90% less than the amount ofinflammation measured in the same assay in the absence the compositionof the present invention or in the presence of a control composition. Inparticular embodiments, the in vitro assay is a mixed lymphocytereaction.

In certain embodiments, a composition of the present invention hasangiogenic activity. In particular embodiments, an injured or diseasedtissue (e.g., an injured or diseased tissue undergoing an inflammatoryresponse) exposed to or contacted with a composition of the presentinvention exhibits increased angiogenesis as compared to when theinjured or diseased tissue is similarly treated but not exposed to orcontacted with the composition of the present invention. In certainembodiments, the amount of angiogenesis in the tissue exposed to orcontacted with the composition of the present invention is increased byat least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 100%, at leastabout 150%, at least about 200%, at least about 300%, at least about400%, or at least about 500%, as compared to the amount of angiogenesiswhen the injured or diseased tissue is not exposed to or contacted withthe composition of the present invention. Angiogenesis may be measuredby means available in the art, including, e.g., the hindlimb ischemiamodel described herein.

In particular embodiments, a composition of the present invention hasangiogenic activity that may be measured in an in vivo or in vitroassay. In certain embodiments, the amount of activity measured in an invitro angiogenesis assay in the presence of a composition of the presentinvention is at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least 90 about %, at least about 100%,at least about 150%, at least about 200%, at least about 300%, at leastabout 400%, or at least about 500% greater than the amount of activitymeasured in the same assay in the absence the composition of the presentinvention or in the presence of a control composition. In particularembodiments, the in vivo assay is a matrigel assay, as described inExample 4. In particular embodiments, the in vitro assay is theendothelial cell migration assay described herein.

In certain embodiments, a composition of the present invention promoteshealing of an injured or diseased tissue; i.e., it has tissue healingactivity. As used herein, “tissue healing activity” of a composition isthe ability of the composition to facilitate improved healing (e.g.,repair or regeneration) of an injured or diseased tissue (e.g., a hardor soft tissue) exposed to the composition as compared to an analogoustissue similarly treated but without exposure to the composition.Improved healing is measured using any appropriate means, such as timeto complete healing, amount of new tissue generated, strength of theresulting healed tissue, or functionality of the resulting healedtissue.

Sterilized or virus-inactivated allogeneic and xenogeneic multipotentcell and processed microvascular tissue compositions have not previouslybeen used to facilitate repair of soft tissues such as ligaments andtendons, because of the difficulty of producing new soft tissue withautologous stem cells, the perception that allogeneic and xenogeneicstem cells will be rejected, and the prior belief that sterilized orvirus-inactivated cells will have reduced viability and, thus, reducedbiological or therapeutic activity. However, the process andcompositions described herein do not necessarily rely on purified stemcells or cell viability. Rather, the provided process is used to producea composition containing a mixture of cells, including nonviable cells,mesenchymal stem and progenitor cells, and other molecules secreted bysuch cells (e.g., cytokines, growth factors, chemotactic molecules, andthe like). In some embodiments, the composition contains a mixture ofviable and nonviable cells.

In particular embodiments, less than about 50%, less than about 40%,less than about 30%, less than about 20%, less than about 10%, or lessthan about 5% of the cells present in a composition of the presentinvention are viable. In several embodiments, substantially all of thecells are non-viable. As used herein, the term “viable” shall be givenits ordinary meaning and shall also refer to a cell that is capable ofproliferating when cultured under appropriate conditions, e.g.,conditions under which the same cell or type of cell would be expectedto proliferate, e.g., if not processed as described herein. In otherembodiments, less than about 2% or less than about 1% of the cellspresent in said composition are viable. In particular embodiments, noneor substantially none of the cells present in the composition areviable. Accordingly, the term “non-viable” means that the cell is notcapable of proliferating when cultured under appropriate conditions,e.g., conditions under which the same cell would be expected toproliferate, e.g., if not processed as described herein.

However, in particular embodiments, at least some of the cells within acomposition of the present invention exclude trypan blue. In particularembodiments, at least about 1%, at least about 2%, at least about 3%, atleast about 5%, at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or at least about 90% of the cellspresent in a composition exclude trypan blue.

In certain embodiments, a composition of the present invention comprisescells that exclude trypan blue but are not viable. In certainembodiments, at least about 1%, at least about 2%, at least about 5%, atleast about 10%, at least about 20%, or at least about 50% of cellspresent within a composition exclude trypan blue but are not viable.

It is understood according to the present invention that, although cellswithin the compositions described herein may not be viable and may notpersist long after being transplanted into a subject, the compositionstrigger a cascade of responses in the subject that lead to improvedhealing, reduced inflammation, or increased angiogenesis. Themultipotent cell and processed microvascular tissue compositionsdescribed herein need not include viable or whole stem cells to promoteor induce healing of injured or diseased tissue, such as, e.g., softtissue. In addition, the compositions of the present invention maycomprise processed tissue and various components thereof, includingdissociated tissue, cells, such as multipotent cells (e.g., stem cells),cell membranes, extracellular matrix components, and various growthfactors, angiogenic factors, anti-inflammatory agents, cytokines,differentiation agents, etc. present within or associated with a tissuesample used to prepare the compositions.

For the purposes of administering a composition of the invention to asubject in need thereof, the compositions may be formulated aspharmaceutical compositions. Pharmaceutical compositions of the presentinvention comprise a composition of the present invention and apharmaceutically acceptable excipient, carrier and/or diluent. Thecomposition of the invention is present in the pharmaceuticalcomposition in an amount sufficient to effect treatment or prevention ofan injury, disease or disorder in a subject in need thereof, i.e., in atherapeutically effective amount.

Pharmaceutically acceptable excipients, carriers and/or diluents arefamiliar to those skilled in the art. For compositions formulated asliquid solutions, acceptable carriers and/or diluents include saline andsterile water, and may optionally include antioxidants, buffers,bacteriostats and other common additives. The pharmaceuticalcompositions of the invention can be prepared by combining a compositionof the invention with an appropriate pharmaceutically acceptablecarrier, diluent or excipient, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as powders,granules, solutions, injections, inhalants, microspheres and aerosols.These compositions may also contain dispersing and surface activeagents, binders and lubricants. One skilled in this art may furtherformulate a composition of the invention in an appropriate manner, andin accordance with accepted practices, such as those disclosed inRemington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co.,Easton, Pa. 1990.

Routes of administering the pharmaceutical compositions of the inventioninclude, without limitation, topically, intramuscular, intravenous,intraarterial, intraperitoneal, subcutaneous, oral, nasal,transplantation, implantation, injection, delivery via a catheter,topical, transdermal, inhalation, parenteral, and intranasal. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. Inaddition, the compositions of the invention may be surgically implanted,injected, delivered (e.g., by way of a catheter or syringe), orotherwise administered directly or indirectly to the site in need ofrepair or augmentation. For example, compositions of the presentinvention may be surgically introduced into or adjacent to a site ofinjury or disease in a subject. In some embodiments, administration isintravenous. Pharmaceutical compositions may be formulated for aparticular route of administration. In particular embodiments, themethod is surgically for tissue repair, intravenously for treatment ofischemia, injection into joint spaces for treatment of pain andinflammation, injection into wounds, and injection into muscle fortreatment of peripheral vascular disease.

In certain embodiments, a composition of the present invention isformulated for intravenous administration. A composition formulated forintravenous administration, in certain embodiments, is filtered toreduce large particles or clumps that could potentially clog capillariesor other blood vessels. In particular embodiments, a compositionformulated for intravenous administration is optionally isotonic, has apH in the range of about 5.0 to about 9.0 (e.g., about 7.3), anosmolarity between about 50 and about 600, and/or an osmolality lessthan or equal to about 600 mOsm/L, e.g., about 290 mOsm/L.

Implants, Matrices and Scaffolds

In certain embodiments, compositions of the present invention arecombined with an implant, matrix or scaffold. Matrices may includebiocompatible scaffolds, lattices, self-assembling structures and thelike. Such matrices are known in the arts of cell-based therapy,surgical repair, tissue engineering, and wound healing. The matrices maybe pretreated (e.g., seeded, inoculated, contacted with) with acomposition of the invention. In some aspects of the invention, cellsand/or tissue components present within the composition adhere to thematrix. In some embodiments, the cells are contained within or bridgeinterstitial spaces of the matrix. In particular embodiments, the cellsand/or other tissue components are in close association with the matrixand, when used therapeutically, induce or support ingrowth of thesubject's cells and/or angiogenesis.

Matrices associated with or comprising compositions of the presentinvention can be introduced into a subject's body in any way known inthe art, including but not limited to implantation, injection, surgicalattachment, transplantation with other tissue, and the like. Acomposition of the present invention may be combined with an implant,matrix or scaffold before being provided to or implanted within asubject, or a composition of the present invention may be combined withan implant, matrix or scaffold already present within a subject.Implants, matrices or scaffolds can provide a physical structure thatretains the composition within a desired location within a subject ortissue therein, protects the composition within the subject, and/orallows release or the composition at a desired rate or over a desiredtime period.

The matrices used in several embodiments may be configured to the shapeand/or size of a tissue or organ in vivo. The scaffolds of the inventionmay be flat or tubular or may comprise sections thereof, as describedherein. The scaffolds of the invention may be multilayered.

In particular embodiments, the implant, matrix or scaffold is abiocompatible implant, matrix, or scaffold. The implant, matrix orscaffold may comprise a solid or liquid. The implant, matrix, orscaffold may be biodegradable. The implant, matrix or scaffold, inparticular embodiments, comprises microbeads or particles, abone-derived implant, a biofiber scaffold (e.g., BioFiber™ Scaffold), aporous resorbable polymer, a hydrogel, a putty comprising tissueproduct, or a suture or an implantable medical device. The implant,matrix or scaffold may be, e.g.: a collagen matrix or biocompatiblefabric; an orthopedic implant; a porous, flexible implantable scaffold;a surgical implant; a porous coated implant; a polymer solution;solvents such as DMSO, N-methylpyrrolidone (NMP), and alcohols; ahydrogel; hyaluronic acid or other glycosaminoglycans or proteoglycans;collagen; fibrin; thrombin; blood clot; platelets; platelet rich plasma;demineralized bone matrix; autologous cells; and/or cancellous bone.

Compositions of the invention may be suspended in a hydrogel solution,e.g., for injection. Examples of suitable hydrogels includeself-assembling peptides, such as RAD16. Alternatively, the hydrogelsolution containing the cells may be allowed to harden to form a matrixhaving cells dispersed therein prior to implantation. The hydrogel maybe an organic polymer (natural or synthetic) that is cross-linked viacovalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure that entraps water molecules to form a gel.Examples of materials that can be used to form a hydrogel includepolysaccharides such as alginate and salts thereof, peptides,polyphosphazines, and polyacrylates, which are crosslinked ionically, orblock polymers such as polyethylene oxide-polypropylene glycol blockcopolymers which are crosslinked by temperature or pH, respectively.

In particular embodiments, a composition of the present invention isassociated with, contained within, applied to, or coating a biologicallycompatible implant, matrix or scaffold. In particular embodiments, acomposition of the present invention is used to coat a material, suchas, e.g., a flexible biocompatible scaffold (e.g., woven or nonwovenfabric sheets or thread). Spray dried processed compositions areparticularly well-suited to coating a material comprising microbeads orparticles without requiring reconstitution prior to coating, as coatingcan be done during the spray drying process. In certain embodiments, thecomposition is embedded within or coated on a matrix, e.g., a porousand/or collagen-containing matrix. In certain embodiments, the matrixmay be Conexa™ reconstructive tissue matrix, BioFiber™ Scaffold, orBioFiber™-CM Scaffold (Tornier; Bloomington, Minn.).

In certain embodiments, compositions of the invention may be associatedwith a three-dimensional scaffold and implanted in vivo, where thecomposition induces cell proliferation on or in the framework and formsa replacement tissue in vivo. The cells that proliferate on or in theframework may include cells within the composition and/or cells of thesubject in whom the scaffold is implanted. Such three-dimensionalframework can be used to form tubular structures, like those of thegastrointestinal and genitourinary tracts, as well as blood vessels;tissues for hernia repair; tendons and ligaments. In relatedembodiments, compositions of the present invention are associated with athree-dimensional framework. The framework may be configured into theshape of the corrective structure desired.

Examples of scaffolds which may be used in the present invention includebut are not limited to nonwoven mats, porous foams, or self-assemblingpeptides, as described, e.g., in U.S. Pat. No. 7,560,276, which isincorporated in its entirety by reference herein. Nonwoven mats may beformed using fibers comprised of a synthetic absorbable copolymer ofglycolic and lactic acids (PGA/PLA)(VICRYL; Ethicon, Inc., Somerville,N.J.) or poly-4-hydroxybutyrate (PHA, Tepha, Lexington, Mass.). Foams,composed of, for example, poly(epsilon-caprolactone)/poly(glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilized, as discussed in U.S. Pat. No. 6,355,699, may also be used.In one embodiment, the framework is a felt, which can be composed of amultifilament yarn made from a bioabsorbable material, e.g., PGA, PLA,PHA, PCL copolymers or blends, or hyaluronic acid.

Kits

The present invention further comprises kits comprising a composition,e.g., a pharmaceutical composition, of the present invention. Thecomposition may be packaged alone, for example, in a vial, or incombination with other products, such as those suitable for combinationwith processed or cryopreserved microvascular tissue. When packaged withanother material, the processed or cryopreserved microvascular tissuecan be separately packaged, or premixed or associated with the othermaterial. In some embodiments, processed microvascular tissue ispackaged as a coating on a biocompatible material, or associated with animplant, matrix or scaffold.

In particular embodiments, a kit of the present invention comprises amoisture impermeable container comprising a composition describedherein. For example, in one embodiment, a kit of the present inventioncomprises a moisture impermeable container comprising a sterile, dried(e.g., lyophilized) composition, wherein said composition comprisesisolated multipotent cells or processed microvascular tissue, or a cellmembrane comprised of said cells or tissue, wherein said cells or tissuehave not been cultured, wherein said composition has angiogenic oranti-inflammatory activity, wherein said composition is sterilizedand/or viruses within said composition are inactivated.

In particular embodiments of kits and compositions described herein, thecomposition retains measurable angiogenic or anti-inflammatory activitywhen stored at approximately room temperature for at least one month, atleast two months, at least four months, at least six months, or at leastone year. In particular embodiments of kits and compositions describedherein, the composition retains measurable angiogenic oranti-inflammatory activity when stored at approximately room temperaturefor at least one month, at least two months, at least four months, atleast six months, or at least one year. As used herein, “roomtemperature” is a temperature of about 20° C. to about 25° C. or about21° C. In particular embodiments of kits and compositions describedherein, the composition retains measurable angiogenic oranti-inflammatory activity when stored at approximately 4° C. for atleast about one month, at least about two months, at least about fourmonths, at least about six months, or at least about one year. Inparticular embodiments of kits and compositions described herein, thecomposition retains measurable angiogenic or anti-inflammatory activitywhen stored at approximately −20° C. for at least one about month, atleast two about months, at least about four months, at least about sixmonths, or at least about one year. In particular embodiments, themeasurable angiogenic or anti-inflammatory activity is at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90% of the activity prior to storage, whenmeasured in an in vivo or in vitro assay, including any of thosedescribed herein.

In particular embodiments of kits and compositions of the presentinvention, less than or equal to 50%, less than or equal to 40%, lessthan or equal to 30%, less than or equal to 20%, less than or equal to10%, or less than or equal to 5% of the cells present in saidcomposition are viable, less than or equal to 2% of the cells present insaid composition are viable, or substantially none of the cells presentin said composition are viable. In related embodiments, at least 1% ofsaid cells exclude trypan blue. In other embodiments, at least about 2%,at least about 5%, at least about 10%, at least about 20%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,or at least about 90% of said cells exclude trypan blue. In certainembodiments, at least about 1%, at least about 2%, at least about 5%, atleast about 10%, at least about 20%, at least about 50%, at least about60%, at least about 70%, at least about 80%, or at least about 90% ofsaid cells exclude trypan blue but are not viable.

According to various embodiments, a kit of the present invention maycomprise a pharmaceutical composition comprising a composition of thepresent invention and an excipient. In one embodiment, thepharmaceutical composition is formulated for intravenous administration.In other embodiments, a kit of the present invention may comprise acomposition of the present invention and an implant, scaffold or matrix,including but not limited to any of those described herein. In specificembodiments, the implantable scaffold or matrix is a bone-derivedimplant, a biofiber scaffold, a porous resorbable polymer, a hydrogel, aputty comprising tissue product, or a suture. In certain embodiments,cells, tissue or cell membrane of said composition are present on abone, tendon or dermal facing surface of said implantable scaffold ormatrix.

In certain embodiments, a kit of the present invention comprises asterilized and dried (e.g., lyophilized) composition of the presentinvention in a sealed container. The sealed container may be moistureresistant or moisture impermeable, and it may contain a sealed opening,allowing access to the interior of the container. In certainembodiments, the container is a vial comprising a hermetic seal. Incertain embodiments, the container is a blister pack, which may comprisea foil seal. In particular embodiments, the interior of the sealedcontainer is sterile. Accordingly, prior to use, the user may access theinterior of the container, add a liquid to the dried composition todissolve or reconstitute it, and then provide or administer thereconstituted composition to a subject. In certain embodiments, theliquid is sterile water or a sterile solution such as saline, e.g.,phosphate buffered saline.

Uses of the Compositions and Implants, Matrices, and Scaffolds

Compositions of the present invention, and implants, matrices, and/orscaffolds comprising said compositions, may be used to treat or preventan injury or disease in a subject in need thereof. In variousembodiments, the compositions may be provided or applied directly to atissue in need thereof or adjacent to a tissue in need thereof, e.g., toa tissue surrounding the tissue in need thereof. Subjects in needthereof include subjects having an injury or disease, or at risk of, aninjury or disease that might benefit from treatment with a compositionof the present invention. In particular embodiments, a subject is amammal, e.g., a human or other mammal, such as a non-human primate, adog, a cat, or a horse. In certain embodiments, a subject has reducedhealing capabilities, such as a diabetic subject and a subjectundergoing chemotherapy.

In certain embodiments, compositions of the present invention are usedto treat or prevent injury or disease of various tissues or organs in asubject, including but not limited to soft tissue injury or disease,hard tissue injury or disease, bone injury or disease, joint injury ordisease, cardiac tissue injury or disease, adipose tissue injury ordisease, cartilage injury or disease, and intervertebral disc injury ordisease.

In certain embodiments, compositions of the present invention are usedto treat or prevent a soft tissue injury. Soft tissue, as used herein,refers generally to extraskeletal structures found throughout the bodyand includes but is not limited to cartilage tissue, meniscal tissue,ligament tissue, tendon tissue, intervertebral disc tissue, periodontaltissue, skin tissue, vascular tissue, muscle tissue, fascia tissue,periosteal tissue, ocular tissue, pericardial tissue, lung tissue,synovial tissue, nerve tissue, brain tissue, kidney tissue, bone marrow,urogenital tissue, intestinal tissue, liver tissue, pancreas tissue,spleen tissue, adipose tissue, and combinations thereof. Soft tissueinjuries include damage or injury to any soft tissue, such as, e.g.,muscles, ligaments, tendons, skin, fibrous tissue, fat, synovialmembranes, nerves, blood vessels, and fascia, which may occur throughoutthe body. Soft tissue injuries that can benefit from the soft tissuehealing activity of the provided processed microvascular tissuesinclude, without limitation, injuries such as tendon and/or ligamenttears and injuries resulting from ischemic events. Common soft tissueinjuries may result from sprain, strain, an injury resulting in acontusion, or overuse of a particular soft tissue. Soft tissue injuriesinclude both open and closed soft tissue injuries.

Soft tissue injuries, disease and conditions that may be treated orprevented according to methods of the present invention include, but arenot limited to, injuries to vascular, skin, or musculoskeletal tissue.Soft tissue conditions include, for example, conditions of skin (e.g.,scar revision or the treatment of traumatic wounds, severe burns, skinulcers (e.g., decubitus (pressure) ulcers, venous ulcers, and diabeticulcers), and surgical wounds such as those associated with the excisionof skin cancers); vascular condition (e.g., vascular disease such asperipheral arterial disease, coronary artery disease, abdominal aorticaneurysm, carotid disease, and venous disease; vascular injury; impropervascular development); conditions affecting vocal cords; cosmeticconditions (e.g., those involving repair, augmentation, orbeautification); muscle diseases (e.g., congenital myopathies;myasthenia gravis; inflammatory, neurogenic, and myogenic musclediseases; and muscular dystrophies such as Duchenne muscular dystrophy,Becker muscular dystrophy, myotonic dystrophy, limb-girdle-musculardystrophy, facioscapulohumeral muscular dystrophy, congenital musculardystrophies, oculopharyngeal muscular dystrophy, distal musculardystrophy, and Emery-Dreifuss muscular dystrophy); conditions ofconnective tissues such as tendons and ligaments, including but notlimited to a periodontal ligament and anterior cruciate ligament; andconditions of organs and/or fascia (e.g., the bladder, intestine, pelvicfloor). One example of a fairly common soft tissue injury is damage tothe pelvic floor. This is a potentially serious medical condition thatmay occur during childbirth or from complications thereof which can leadto damage to the vesicovaginal fascia, such as a cystocele, which is aherniation of the bladder. Similar medical conditions include rectoceles(a herniation of the rectum), enteroceles (a protrusion of the intestinethrough the rectovaginal or vesicovaginal pouch), and enterocystoceles(a double hernia in which both the bladder and intestine protrude).

In various embodiments, compositions of the present invention are usedto treat or prevent various diseases, including but not limited todiseases associated with undesirable inflammatory or immune responses.Examples of such disease include rheumatoid arthritis, osteoarthritis,and autoimmune diseases and disorders. In addition, compositions of thepresent invention may be used to reduce inflammation, e.g., at a site ofinjury, and/or to reduce an immune response, e.g., an immune responseinduced by an injury. Similarly, compositions of the present inventionmay be used to prevent or reduce the likelihood of transplant rejection.

In particular embodiments, compositions of the present invention areused to promote or stimulate angiogenesis or revascularization, e.g., ata site of injury or tissue damage. In particular embodiments, the injuryis associated with or resulted in ischemia, hypoxia, or reperfusioninjury to a tissue. Examples of injuries or diseases associated with orresulting in ischemia, hypoxia, or reperfusion injury that may betreated or prevented according to the present invention include stroke,myocardial infarct, and blood loss. Additional examples of injuries ortissue damage that may be treated with compositions of the presentinvention to promote or stimulate angiogenesis or revascularizationinclude transplantation or limb reattachment.

Compositions of the present invention may also be used to treat orprevent peripheral nerve damage, erectile dysfunction, pulmonaryhypertension, multiple sclerosis, and radiation burns. In addition, theymay be used to induce hematopoiesis and/or wound healing.

In particular embodiments, compositions of the present invention, e.g.,when formulated for intravenous administration, may be used to treat orprevent acute myocardial infarct, congestive heart failure, stroke,peripheral vascular disease or chronic obstructive pulmonary disease.

The compositions of the present invention may be used alone or incombination with one or more other therapeutic agents or procedures totreat or prevent an injury or disease. For example, in certainembodiments, to enrich blood supply to a damaged tissue and/or topromote tissue regeneration, compositions of the present invention maybe used in combination with platelet-rich plasma. When used incombination with one or more other therapeutic agents or procedures, thecompositions of the present invention may be provided or used prior to,at the same or during an overlapping time period as, or subsequent to,treatment with the other therapeutic agent or procedure.

When used in combination with another therapeutic agent, a compositionof the present invention may be provided separately from the otheragent, or it may be present in a pharmaceutical composition that alsocontains the other therapeutic agent, e.g., a coformulation comprisingtwo or more therapeutic agents, one being the composition of the presentinvention. In particular embodiments, the composition of the presentinvention and an additional therapeutic agent are both combined with orassociated with the same implant, matrix or scaffold.

The compositions of the invention are administered in a therapeuticallyeffective amount, which will vary depending upon a variety of factorsincluding the activity of the specific composition employed; the age,body weight, general health, sex and diet of the subject to which thecomposition of the invention is administered; the mode and time ofadministration; the rate of excretion or breakdown of the composition inthe subject; and the type or severity of the injury, disease, orcondition to be treated. In certain embodiments, a therapeuticallyeffective dose results from the material obtained by processing 10⁴ to10⁸ multipotent cells and their associated ECM.

The methods of the present invention may be practiced by administeringthe composition in one, two or more doses. For example, in certainembodiments, a composition is administered as a single dose, multipledoses or in repeated doses over a period of time.

EXAMPLES Example 1—Microvascular Tissue Preparation And Characterization

In this study, microvascular tissue is prepared via different processesand then assayed. In brief, at least 5 to 10 lbs of subcutaneous fat isobtained from an organ donor and processed as follows: mince the tissue;enzymatically dissociate it; dilute (no quench), spin, decant and thenwash the cell pellet; resuspend in lyophilization buffer; andlyophilization. The base conditions used for processing are as follows:adipose tissue is surgically recovered from a tissue donor. The tissueis minced with scissors, suspended in PBS with 0.2 U/ml CIzyme AS(Vitacyte, Indianapolis, Ind.) at 37° C. with gentle agitation for 60min, then washed three times and resuspended at two million cells/ml inM3D. The cell suspension is held at room temperature until just prior tolyophilization, when the cells are diluted 50:50 with EZ-CPZ™cryopreservation media (Incell Corp., San Antonio, Tex.), vialed, andloaded into the lyophilizer trays for cooling. The samples are assayedboth during the process and at the end of the process.

10 process methods are performed in this study. They are designated “A”through “J” in Table 1. The assay methods used to analyze each processmethod are listed in Table 1, designated 1, 2, 3, 4, 5, and detailed inTable 2.

TABLE 1 Process Methods, Descriptions and Assay Methods Process *AssayMethod Description of Process Method Method(s) A Control: 10 gm of fatin ZTM ™ minced with M2, 3, 4, 5 scissors/scalpel immediately (<12 hrfrom harvest) and digested according to base conditions. B 24 hr: 10 gmof fat in ZTM ™ stored at room temperature M2, 4, 5 for 24 hr beforeprocessing with scissors/scalpel and base conditions. C 48 hr: 10 gm offat in ZTM ™ stored at room temperature M2, 4, 5 for 48 hr beforeprocessing with scissors/scalpel and base conditions. D Scale-up: 5+pounds of fat minced using a meat grinder; M1, 2, 3, 4 digested per baseconditions. E Enzyme: 10 gm of minced fat from ‘D’ digested with 4X M2,5 the enzyme concentration. F Volume: 10 gm of minced fat from ‘D’digested with 4X M2, 5 the enzyme concentration but in ¼ the totalvolume of ‘E’. G Lyophilization: Use formulation of 50:50 EZ- M2, 3, 4,5 CPZ ™:M3D ™ on all of ‘D’ product. The shelf was cooled from roomtemperature to −45 C. at a cooling rate of 2.5 C./min until producttemperature reached −45 C. Samples were maintained −45 C. for 180minutes. Primary drying was initiated by raising the shelf temperaturefrom −45 C. to −35 C. at 2.5 C./minute, reducing the chamber pressure to80 mTorr and maintaining the shelf temperature at −35 C. for 2160minutes. For secondary drying, samples were warmed by increasing theshelf temperature at a rate of 0.2 C./minute to 20 C. and maintainingthe shelf at this temperature for 360 minutes. H Sterilization: 2.5 Mradof gamma radiation of 100 vials M2, 3, 5 of ‘G’. Use for stability. ILow Dose: 1.5 Mrad of gamma radiation of 80 vials of M2, 3, 5 ‘G’. Usefor animal studies, along with 200 vials unsterilized. J E-Beam: 1.5Mrad of e-beam radiation of 100 vials of M2, 3, 5 ‘G’. Use forstability.

TABLE 2 Study Assay Groups Assay Group Description M1 Donor screeningfor infectious diseases including tests not done prior to fat tissueharvest. M2 Cell counts using a hemacytometer and Trypan Blue with DAPInuclear staining) for cell number and viability. M3 Immunophenotypingfor selected Biomarkers: CD33, CD34, CD44, CD45, and collagen Type IV M4Bioburden M5 Functional Bioassays: Cell migration; Matrigel

Several tasks are performed for this study. They are designated A to Dand described in further detail below and outlined in FIG. 1. Thespecific laboratory assay methods (M) used in the Tasks are designatedas M1, M2, M3, M4 and M5 (Table 2).

Task A. Planning and Set-Up

Order materials, coordinate, set-up and testing.

Task B. Source Materials and General Procedures

The fat tissue is obtained from an organ donor. Subcutaneous fat istaken from abdomen, thighs and buttocks. Five (5) to ten (10) pounds offat are harvested into ZTM™ transport medium (Incell Corp., San Antonio,Tex.).

The tissue is harvested and initially processed within 12 hours of deaththrough various steps and laboratory methods (Tables 1 and 2; FIG. 1).Ten (10) gm aliquots are processed using base conditions after 12, 24and 48 hours storage at room temperature. The >5+ pounds is processedusing a meat grinder method for tissue mincing, and 10 gm aliquots aredigested with 4× the Blendzyme I (Vitacyte) enzyme concentration inZSolM™ and with 4× enzyme concentration and ¼ the digestion volume ofZSolM™ in Step E. The bulk of the sample is digested at the standardenzyme concentrations and methods.

After digestion, the samples are rinsed in ZSolF™, centrifuged,decanted, then washed two more spins in ZSolF™. The cells areresuspended 1:1 cell suspension in EZ-CPZ™ as a lyophilization solutionbulk product at 10⁶ cells/ml and lyophilized (Step G) as 1 mL aliquotvolumes. Lyophilized vials are subjected to gamma and E-beam irradiation(Steps H, I, J). All end-product is stored and representative samplesare tested, and selected subsets are used in subsequent animal studies.

Task C. Assays

The various types of assays (M1 to M5) used in this study (Table 2) arebriefly summarized below.

M1: Donor screening for infectious diseases including tests not doneprior to fat tissue harvest. Donor screening and agreements for tissueprocurement are developed to minimize or eliminate any infectiousdiseases according to the standard evaluations. Actual additional testsand associated costs are performed on a case-by-case basis. Bioburdenassays are performed.

M2: Cell counts are performed using a hemacytometer (light microscopy)and Trypan Blue with DAPI nuclear (fluorescence microscopy) staining forcell number and viability. Cell counts are recorded as duplicatereadings.

M3: Immunophenotyping for selected Biomarkers: CD33, CD34, CD44, CD45,and collagen TypeIV. Immediate immunoassays of suspended cells areperformed. Cells are grown out in LabTeks then stained, andrepresentative photos are taken.

M4: Bioburden assays are done on samples from transport solutions ofreceived tissues in bag N=2 (and compared to post-processing {last}rinses. Endotoxin testing is performed using EndoSafe PTS Assay (CharlesRiver). Standard USP culture microbiological testing is performed. ATPrapid testing is optionally performed.

M5: Functional bioassays are done on samples of isolated cells,post-lyophilization, and after the various processing and irradiationprotocols. Cell migration assays across transwells are performed forADSCs, endothelial, fibroblasts. Matrigel assays are performed toevaluate microvessel formation induced by samples at various dilutions,stages of processing and/or irradiation.

Task D. Data Analyses

Observational readings and data are transferred to Excel or to Prism foranalysis. The mean+SD values of sample replicates for each TPS and eachcell type are tabulated and/or plotted for comparative analyses.

The results obtained from preliminary experiments that were performedshowed little difference between processing conditions A, B and D with3.5 kg of adipose tissue generating 1560 vials of lyophilized product at106 cells per vial.

Example 2—Treatment of Achilles Tendon Injury in Rats

This study demonstrates that microvascular tissue preparations of thepresent invention can be used to repair Achilles tendon injuries.

32 male Sprague Dawley rats (8 weeks old on DAY 1 and ˜250 g on DAY 1)are purchased from Harlan and acclimatized for at least 3 days. The ratsare treated as summarized in the study design of Table 3.

TABLE 3 Study Design # Animal/ Group group Treatment Route Endpoints 1 8Achilles tendon is slightly Right Surgery- implant abraded withmouse-tooth Tibia scaffold between forceps tibia and 2 8 Achilles tendonis slightly Achilles tendon abraded with mouse-tooth Terminationforceps + Tornier's Graft Day 7 Material coated with Collagen 3 8Achilles tendon is slightly abraded with mouse-tooth forceps + Tornier'sGraft Material coated with Collagen + Processed microvascular tissuecomposition A 4 8 Achilles tendon is slightly abraded with mouse-toothforceps + Tornier's Graft Material coated with Collagen + Processedmicrovascular tissue composition B TOTAL 32

The study occurs over approximately 10 days of animal life. The animalarrives on day −3, is acclimatized day −3 to day 1, subjected to surgeryon day 1, and scheduled for termination on day 7.

Test Articles:

Collagen Coated BioFiber Scaffolds

Processed microvascular tissue preparations A and B reconstituted withsterile WFI and absorbed into scaffolds. Processed microvascular tissuecomposition A is unsterilized, and processed microvascular tissuecomposition B is E-beam sterilized.

Anesthesia:

Prior to surgery on Day 1, animals are weighed and anesthetized with anintramuscular injection of ketamine hydrochloride 100 mg/mL (40-mg/kg)and xylazine 100 mg/mL (5-10 mg/kg).

Surgical Preparation:

The right rear limb of each animal is shaved one day prior to the startof the test. On Day 1, the skin is surgically prepared with betadine andalcohol scrubs, and draped using aseptic surgical techniques.

Surgical Procedure:

On Day 1, the test article is prepared immediately prior toimplantation. The graft is loaded with cells by wicking action. Two 5-0polypropylene sutures are placed in the graft for fixation. The graft isset aside in the Petri dish with saline and covered until used.

A straight, lateral skin incision is made from the caudal (distal) tibiaof the right rear limb to the level of the mid tibia. Using this method,the skin is dissected and retracted to allow a lateral exposure of theAchilles tendon from calcaneus to its musculo-tendinous junction.Further dissection is used to expose and isolate the Achilles tendon.The exposed Achilles tendon is slightly abraded with mouse-tooth forcepsprior to graft test article placement. A single 0.5 mm drill hole ismade in the lateral to medial direction through the Calcaneus to allowsuture passage for graft fixation. The implant area is irrigated withsaline to remove any debris and blotted dry.

The graft is removed from the holding media and inserted along theanterior surface of the Achilles tendon with one end adjacent to thecalcaneus. The cranial graft fixation suture is placed in thegastrocnemius cranial to the musculo-tendinous junction using a modifiedMason-Allen suture pattern. The caudal graft fixation suture is thenpassed through the drill hole in the calcaneus and tensioned with thefoot in a neutral position and tied. Six suture knots are tied for allfixation sutures. The incision is closed in a layered fashion usingappropriate suture material.

Analgesia:

Animals are administered buprenorphine (0.1-0.5 mg/kg) subcutaneouslyupon recovery from anesthesia on Day 1. Additional buprenorphine may beadministered discretionarily as needed for pain.

Body Weight Measurement:

Animals are weighed at randomization, prior to surgery on Day 1, andonce a week until end of study, including prior to termination. (˜9 timepoints)

Health Observations:

Animals are monitored once daily for the duration of the study. (˜7 timepoints)

Incision site area observations:

Observations of the incision site are recorded daily from Day 2 throughDay 7.

Temperature/Humidity Recordings:

Daily room temperature and humidity measurements are recorded.

Termination and Tissue Collection:

On Day 7, animals are euthanized and the implanted test or controlarticle sites and surrounding tendinous tissue are collected by excisionfrom each animal. All collected samples are split in half along themid-line of the scaffold with half the tendon included in each half.One-half of the collected tissue is stored in 10% neutral bufferedformalin for routine histopathological and immunohistochemistryevaluation. The remaining half is stripped of tendon and overgrown softtissue with the edge of a scalpel, and the scaffold with ingrown tissueis snap frozen at ≤−70° C. in liquid nitrogen for gene expressionanalysis.

Sections of tendon (taken from the contralateral Achilles), skin (takenfrom a region with less fur) and liver of 2 animals/group randomlyselected are also collected as staining controls and stored in 10%neutral buffered formalin for immunohistochemistry evaluation. A portionof each control tissue is snap frozen in liquid nitrogen for PCRcontrols. (All three control tissues can be stored together in formalinand the frozen portions can likewise be stored together).

The tissues are subjected to histology (H&E, Masson's trichrome),immunohistochemistry (SMAD8 and tenascin), and PCR (SMAD8, tenascin,tenomodulin, and scleraxis) analysis.

Example 3—Treatment of Ischemia in Mice

This study demonstrates the effects of processed microvascular tissuecompositions of the present invention in a murine model of limbischemia. The murine model of limb ischemia is created as previouslydescribed (Jang J et al, Circulation 1999; Huang N et al, JOVE 2009),and is used to assess the effects of cell preparations in promotingangiogenesis after induction of hindlimb ischemia.

SCID mice 14-16 weeks old undergo surgically induced hindlimb ischemia.Immediately after surgery, processed microvascular tissue composition orvehicle control will be administered to the animals as detailed in Table4 below. Briefly, three test cell articles (each at 0.5×10⁶ cells) orvehicle control are injected intramuscularly on day 0 after induction ofhindlimb ischemia into the gastrocnemius. The three test articlesinclude: processed microvascular tissue composition (Test Cells-I),processed microvascular tissue composition sterilized by E-beam (TestCells-II), and processed microvascular tissue composition sterilized bygamma radiation (Test Cells-III). Improvement in limb perfusion isevaluated every 3-4 days for a total of 14 days. After 14 days, theanimals are euthanized. Both the ischemic and contralateralgastrocnemius are explanted and subjected to histological analysis.

TABLE 4 Study Protocol Group # animals Test Material 1 10 Vehicle 2 10Test Cells- I (0.5 × 10⁶) 3 10 Test Cells- II (0.5 × 10⁶) 4 10 TestCells- III (0.5 × 10⁶)

Endpoint Testing

Bloodflow is evaluated by Laser Doppler Imaging at Days 0, 3, 7, 11 and14.

Animals are sacrificed at Day 14, and hindlimb tissue is harvested,processed and stored for explant studies.

Example 4—Vessel Formation in SCID Mice

These studies utilize matrigel plug assays to demonstrate the ability ofthe microvascular tissue preparations of the present invention to formvascular structures in vivo. The matrigel plug assay is a definitiveassay of true vessel formation in vivo. This assay involves implantationof therapeutic cells with matrigel subcutaneously into the abdominalregion. During the course of 2 weeks, the cells within the matrigel arein a favorable environment to form neovessels, some of which mayanastamose with host vessels.

In this assay, 0.5×10⁶ cells are embedded in 0.5 ml matrigelsupplemented with 200 ng/ml basic fibroblast growth factor and theninjected subcutaneously into SCID mice. 2 plugs are implanted into eachanimal. After 2 weeks, the plugs are explanted for histological analysisof vessel formation. To distinguish human from native murine vessels,human specific antibodies targeting endothelial cells (e.g., CD31) areused to identify human-specific vessels. The presence of human specificvessels, as demonstrated histologically by luminal structures performedwith blood elements, is demonstrative of functional endothelial cells.Similarly, mouse-specific endothelial cell antibodies can be used toidentify mouse-specific vessels. The ability of therapeutic cells tosecrete paracrine angiogenic factors results in a relative enhancementin murine vessel formation.

SCID mice 14-16 weeks old undergo implantation of matrigel plugscontaining microvascular tissue preparations of the present invention orvehicle control, as detailed in Table 5 below.

TABLE 5 Study Protocol Group # animals Test Material 1 5 Vehicle 2 5Test Cells- I (0.5 × 10⁶) 3 5 Test Cells- II (0.5 × 10⁶) 4 5 Test Cells-III (0.5 × 10⁶)

The three test articles include: processed microvascular tissuecomposition (Test Cells-I), processed microvascular tissue compositionsterilized by E-beam (Test Cells-II), and processed microvascular tissuecomposition sterilized by gamma radiation (Test Cells-III).

Example 5—Treatment of Rheumatoid Arthritis in Rats

This study demonstrates the efficacy of microvascular tissuepreparations of the present invention in inhibiting the inflammation,cartilage destruction and bone resorption associated with 7 dayestablished type II collagen arthritis in rats.

Test System

-   -   Number of animals: 44    -   Species/Strain or Breed: Lewis rats    -   Vendor: Charles River    -   Age/Wt at Arrival: 125-150 g    -   Gender: Female    -   Age Range at Study Initiation: At least 125 grams at time of        first immunization.    -   Acclimation: Acclimated for 4-8 days after arrival at BBP.    -   Housing: 3-5/animals/cage

Materials

Test articles (Processed microvascular tissue composition preparations)and appropriate vehicle. Triamcinolone and sterile saline for dilution(BBP), Bovine Type II collagen (Elastin Products), Freund's incompleteadjuvant (Difco).

General Study Design

Rats are anesthetized with Isoflurane and given 300 μl of Bovine Type IIcollagen in Freund's incomplete adjuvant injections ID/SC spread overthe distal back, 100 μls per site on Day 0 and again on day 6.

Randomization into each group occurs on day 1 of arthritis (study day10) when disease is obvious in both hind paws (knees will generally havedisease of similar severity to ankles but are difficult to reliablycaliper so the ankle measure is a surrogate for the knee for purposes ofdetermining disease onset). This becomes day 1 of arthritis. Animalswith arthritis are randomized into treatment groups with approximatemean ankle caliper measures for each group.

Treatment (IA, bilateral into both knees) occurs on arthritis day 1only. Knees are treated, because ankles are too small to inject.Systemic effects of treatment are monitored by caliper measures ofankles and local effects of treatment are determined by histopathologyon the knees. Ankle caliper measures are taken daily from days 0(baseline)-7. Baseline ankle caliper measurements are taken on day 0using one ankle with values rounded to one-thousandth of an inch.Measurements are confirmed as clinically normal (0.260-0.264 in) bycomparison with historical values for rats based on a range of bodyweights. Baseline measurements are then applied to both ankles, andthese values remain with the animal so long as the ankle is clinicallynormal with good definition of all the ankle bones and no evidence ofinflammation.

TABLE 6 Study Group Designations Group N Compound Route Regimen DoseLevel Dose Vol Dose Conc Grp 1 4 Naïve N/A N/A N/A N/A N/A Grp 2 VehicleControl IA Bilateral, D 1 40 μl Grp 3 8 Triamcinolone IA Bilateral, D 10.03 mgs 40 μl 0.75 mg/ml Grp 4 8 TX-1 PO Bilateral, D 1 2 × 10⁵ cells40 μl 5 × 10⁶ cells/ml Grp 5 8 TX-2 PO Bilateral, D 1 2 × 10⁵ cells 40μl 5 × 10⁶ cells/ml Grp 6 8 TX-3 PO Bilateral, D 1 2 × 10⁵ cells 40 μl 5× 10⁶ cells/ml

The three test articles include: processed microvascular tissuecomposition (TX-I), processed microvascular tissue compositionsterilized by E-beam (TX-II), and processed microvascular tissuecomposition sterilized by gamma radiation (TX-III).

Disease Induction

Acclimated animals are anesthetized with Isoflurane and given collageninjections (D0). On day 6, they are anesthetized again for the secondcollagen injection. Collagen is prepared by making a 4 mg/ml solution in0.01N Acetic acid. Equal volumes of collagen and Freund's incompleteadjuvant are emulsified by hand mixing until a bead of this materialholds its form when placed in water. Each animal gets 300 μl of themixture each time spread over 3 subcutaneous sites (100 μl per site) onback.

Materials

-   -   Name (Vendor): Type II Collagen (Elastin Products)    -   Designation: Bovine Type II Collagen    -   Characteristics: Soluble, from new born calf joints    -   Storage Conditions: 2-8° C.    -   Purity: >99.6%    -   Name (Vendor): Freund's Incomplete adjuvant (Difco)    -   Designation: Incomplete Adjuvant    -   Storage Conditions: 2-8° C. Test

Article and Vehicle

Test Article Vehicle: Stem cell preparations prepared on the day ofinjection, Triamcinolone (Vetalog, Ft. Dodge).

Test Article and Formulation: Stem cell preparations in physiologicvehicle at concentrations appropriate for injecting 40 μl/knee joint.Triamcinolone 2 mg/ml to be diluted in saline.

TABLE 7 Study Calendar Day −8 Distribute rats on arrival into groups foracclimation Day 0 Day 1 Day 2 Day 3 Day 4 Anesthetize, 1st CollagenInjection Day 5 Day 6 Day 7 Day 8 Day 9 (0) Day 10 (1) Day 11 (2) 2ndCollagen Weigh, Weigh, Dose, Weigh, Injection Caliper Caliper CaliperBaseline (Enroll) Day 12 (3) Day 13 (4) Day 14 (5) Day 15 (6) Day 16 (7)Day 17 Day 18 Weigh, Weigh, Weigh, Weigh, Weigh, Caliper Caliper CaliperCaliper Caliper Necropsy

Live Phase Conduct

Randomization into each group by arthritis severity is done on arthritisday 1 (study day 10). Treatment (bilateral IA, 40 μl/joint) is initiatedafter randomization (day 1). Body weights and ankle calipermeasures/scores are taken daily.

Humane Practice: Animals showing signs of morbidity according to theBolder BioPATH IACUC Program of Veterinary Care, including the loss ofmore than 20% body weight (within one week) are removed from the studyand humanly sacrificed via CO₂ inhalation.

TABLE 8 Live Phase Deliverables Live Phase Data Collection: Body WeightArthritis days 0-7 Caliper Measure Arthritis days 0-7 Left & RightAnkles Live Phase (Non PK) Sample Collection N/A N/A N/A

Necropsy

Animals are sacrificed on Arthritis Day 7 by exsanguination.

Necropsy data is collected, including the weight of both the left andright hind paws, and the weight of the liver, spleen and thymus.

Necropsy samples collected include an aliquot of term serum, and theleft and right hind paws and kneed.

Processing of Joints/Histopathologic Scoring

Following 1-2 days in fixative and then 4-5 days in decalcifier, theankle joints are cut in half longitudinally, knees are cut in half inthe frontal plane, processed, embedded, sectioned and stained withtoluidine blue.

Histopathologic Scoring Methods for Rats Joints with Type II CollagenArthritis

Collagen arthritic ankles and knees are given scores of 0-5 forinflammation, pannus formation and bone resorption according to thefollowing criteria:

-   -   Knee and/or Ankle Inflammation        -   0=Normal        -   0.5=Minimal focal inflammation        -   1=Minimal infiltration of inflammatory cells in            synovium/periarticular tissue.        -   2=Mild infiltration        -   3=Moderate infiltration with moderate edema.        -   4=Marked infiltration with marked edema        -   5=Severe infiltration with severe edema

The inflammatory infiltrate in mice and rats with type II collagenarthritis consists of neutrophils and macrophages with smaller numbersof lymphocytes when the lesions are in the acute to subcute phase.Tissue edema and neutrophil exudates within the joint space are commonin the acute to subacute phase. As the inflammation progresses tochronic, mononuclear inflammatory cells (monocytes, lymphocytes)predominate and fibroblast proliferation, often with deposition ofmetachromatic matrix, occurs in synovium and periarticular tissue.Exudate is less common in the joint space. Unless indicated in thecomments area, the inflammation type is acute to subacute.

Ankle Pannus

-   -   0=Normal    -   0.5=Minimal infiltration of pannus in cartilage and subchondral        bone, affects only marginal zones and only a few joints.    -   1=Minimal infiltration of pannus in cartilage and subchondral        bone, primarily affects marginal zones.    -   2=Mild infiltration (<¼ of tibia or tarsals at marginal zones)    -   3=Moderate infiltration (¼ to ⅓ of tibia or small tarsals        affected at marginal zones)    -   4=Marked infiltration (½-¾ of tibia or tarsals affected at        marginal zones)    -   5=Severe infiltration (>¾ of tibia or tarsals affected at        marginal zones, severe distortion of overall architecture)

Knee Pannus

-   -   0=Normal    -   0.5=Minimal infiltration of pannus in cartilage and subchondral        bone, affects only marginal zones and only a few joints.    -   1=Minimal infiltration of pannus in cartilage and subchondral        bone, approximately 1-10% of cartilage surface or subchondral        bone affected.    -   2=Mild infiltration (extends over up tot/4 of surface or        subchondral area of tibia or femur), approximately 11-25% of        cartilage surface or subchondral bone affected    -   3=Moderate infiltration (extends over >¼ but <½ of surface or        subchondral area of tibia or femur) approximately 26-50% of        cartilage surface or subchondral bone affected    -   4=Marked infiltration (extends over ½ to ¾ of tibial or femoral        surface) approximately 51-75% of cartilage surface or        subchondral bone affected    -   5=Severe infiltration approximately 76-100% of cartilage surface        or subchondral bone affected

Ankle Cartilage Damage (Emphasis on small tarsals)

-   -   0=Normal    -   0.5=Minimal decrease in T blue staining, affects only marginal        zones and affects only a few joints    -   1=Minimal to mild loss of toluidine blue staining with no        obvious chondrocyte loss or collagen disruption    -   2=Mild loss of toluidine blue staining with focal mild        (superficial) chondrocyte loss and/or collagen disruption    -   3=Moderate loss of toluidine blue staining with multifocal        moderate (depth to middle zone) chondrocyte loss and/or collagen        disruption, smaller tarsals affected to ½-¾ depth with rare        areas of full thickness loss    -   4=Marked loss of toluidine blue staining with multifocal marked        (depth to deep zone) chondrocyte loss and/or collagen        disruption, 1 or 2 small tarsals surfaces have full thickness        loss of cartilage    -   5=Severe diffuse loss of toluidine blue staining with multifocal        severe (depth to tide mark) chondrocyte loss and/or collagen        disruption affecting more than 2 cartilage surfaces

Knee Cartilage Damage

-   -   0=Normal    -   0.5=Minimal decrease in T blue staining, affects only marginal        zones    -   1=Minimal to mild loss of toluidine blue staining with no        obvious chondrocyte loss or collagen disruption    -   2=Mild loss of toluidine blue staining with focal mild        (superficial) chondrocyte loss and/or collagen disruption, may        have few small areas of 50% depth of cartilage affected    -   3=Moderate loss of toluidine blue staining with multifocal to        diffuse moderate (depth to middle zone) chondrocyte loss and/or        collagen disruption, may have 1-2 small areas of full thickness        loss affecting less than ¼ of the total width of a surface and        not more than 25% of the total width of all surfaces    -   4=Marked loss of toluidine blue staining with multifocal to        diffuse marked (depth to deep zone) chondrocyte loss and/or        collagen disruption or 1 surface with near total loss and        partial loss on others, total overall loss less than 50% of        width of all surfaces combined    -   5=Severe diffuse loss of toluidine blue staining with multifocal        severe (depth to tide mark) chondrocyte loss and/or collagen        disruption on both femurs and/or tibias, total overall loss        greater than 50% of width of all surfaces combined

Ankle Bone Resorption

-   -   0=Normal    -   0.5=Minimal resorption affects only marginal zones and affects        only a few joints    -   1=Small areas of resorption, not readily apparent on low        magnification, rare osteoclasts    -   2=Mild=more numerous areas of resorption, not readily apparent        on low magnification, osteoclasts more numerous, <¼ of tibia or        tarsals at marginal zones resorbed    -   3=Moderate=obvious resorption of medullary trabecular and        cortical bone without full thickness defects in cortex, loss of        some medullary trabeculae, lesion apparent on low magnification,        osteoclasts more numerous, ¼ to ⅓ of tibia or tarsals affected        at marginal zones    -   4=Marked=Full thickness defects in cortical bone, often with        distortion of profile of remaining cortical surface, marked loss        of medullary bone, numerous osteoclasts, ½-¾ of tibia or tarsals        affected at marginal zones    -   5=Severe=Full thickness defects in cortical bone, often with        distortion of profile of remaining cortical surface, marked loss        of medullary bone, numerous osteoclasts, >¾ of tibia or tarsals        affected at marginal zones, severe distortion of overall        architecture

Knee Bone Resorption

-   -   0=Normal    -   0.5=Minimal resorption affects only marginal zones    -   1=Minimal=small areas of resorption, not readily apparent on low        magnification, approximately 1-10% of total joint width of        subchondral bone affected    -   2=Mild=more numerous areas of resorption, definite loss of        subchondral bone, approximately 11-25% of total joint width of        subchondral bone affected    -   3=Moderate=obvious resorption of subchondral bone approximately        26-50% of total joint width of subchondral bone affected    -   4=Marked=obvious resorption of subchondral bone approximately        51-75% of total joint width of subchondral bone affected    -   5=Severe=distortion of entire joint due to destruction        approximately 76-100% of total joint width of subchondral bone        affected

Periarticular Matrix Deposition (Only scored if an increase is seen inany treated group relative to disease controls)

-   -   0=Normal    -   1=Faint, multi-focal metachromatic staining, no excessive        expansion of periarticular tissue    -   2=Darker, diffuse metachromatic staining, no excessive expansion        of periarticular tissue    -   3=Darker, diffuse metachromatic staining, mild expansion of        periarticular tissue    -   4=Darker, diffuse metachromatic staining, moderate expansion of        periarticular tissue    -   5=Darker, diffuse metachromatic staining, severe expansion of        periarticular tissue

Statistical Analysis

Clinical Data

Data are analyzed using a Student's t-test or Mann-Whitney U test(non-parametric). If applicable, data are further analyzed across allgroups using a one-way analysis of variance (1-way ANOVA) orKruskal-Wallis test (non-parametric), along with the appropriatemultiple comparison post-test. Unless indicated, statistical analysis isperformed on raw (untransformed) data only. Statistical tests makecertain assumptions regarding the data's normality and homogeneity ofvariance, and further analysis may be required if testing resulted inviolations of these assumptions. Significance for all tests is set atp≤0.05.

Percent inhibition of paw weight and AUC is calculated using thefollowing formula:

% Inhibition=A−B/A×100

A=Mean Disease Control−Mean Normal

B=Mean Treated−Mean Normal

Example 6—Treatment of Bone Voids, Cartilage Defects and MeniscalLesions in Goats

This study demonstrates the effect of the microvascular tissuepreparation of the present invention in the treating bone voids,cartilage defects and meniscal lesions. In this study, for each animal,the right rear stifle joint is operated on, and 3 distinct and separatesurgical defects are created. Each right femur tested will have one 8 mmdiameter by 20 mm deep bone defect made in the lateral epicondylarregion of the femur, one 4×7 mm rectangular by 2 mm deep cartilagedefect created in the trochlear sulcus, and one 7 mm long by 1-2 mm widefull thickness meniscal defect created in the white-white zone of themedial meniscus. The treatment group of 3 animals has each defecttreated with the microvascular tissue preparation, while the controlgroup has the defects filled with just the scaffold. The goats areevaluated at 8 weeks to evaluate and characterize the repaired tissue inthe various defect sites. It is expected that the treated defects willbe superior in gross and histologic appearance compared to the controls.

The goat was chosen because of the large relative stifle joint size,ease of handling, use in other cartilage, meniscal and bone repairstudies, and similarity of response to that seen in the human. Variousspecies of goats have been used for cartilage research due to theirlarge joint size, similarity of meniscal repair physiology, thickness ofcartilage in the knee (Stifle) joint between 1.5-2 mm, similar to horsesand humans, and that they possess cancellous and cortical bone similarto humans (secondary osteonal). The bone, cartilage and meniscal repairprocesses are a very complex process which cannot be mimicked in an invitro setting. Animal models are necessary as the physiology of joints,especially injured joints, is very complex and cannot be duplicated inthe laboratory. The animals used in this study are summarized in Table9.

TABLE 9 Animal Use # Housed Common Num- Simulta- Housing Name ber AgeWeight Sex neously Duration Nubian 6 2-4 >120 lb. Cas- 6 6 @ 12 weeksBoer- yo trated Cross Goat Male Nubian 2 2-4 >120 lb. Cas- 2 ReplacementBoer- yo trated animals; Cross Goat Male maximum of 12 weeks

One unicortical epicondylar 8 mm diameter by 20 mm deep defect iscreated in the lateral epicondylar region of the right femur; one 4×7 mmby approximately 2 mm deep rectangular defect is created in the lateraltrochlear sulcus of the right femur, and one approximate 7 mm long by1-2 mm wide full thickness defect will be created in the right medialmeniscus. Each knee is physically examined for drawer, range of motion(goniometer), swelling, temperature, crepitus, patella tracking, andvalgus/varus. A standard surgical scrub with chlorohexidine followed by70% alcohol and followed with a paint of betadine is performed. Thesurgical approach consists of a curved, medial skin incision made fromthe distal one-third of the right femur to the level of the tibialplateau.

The medial collateral ligament is identified and an outline of thebone-ligament attachment footprint is made with cautery. In the centerof the footprint, a 2.8 mm drill bit and tap is used to create a screwhole for reattachment of the ligament. An oscillating saw is used to cutthe attachment footprint of the medial collateral ligament, allowing forit to be reflected towards the tibia. The joint capsule is opened, andthe knee is flexed and rotated laterally to expose the medial meniscus.A plastic protective tab is placed under the medial meniscus and using aspecially designed oval punch, a full thickness defect is made in thewhite-white zone of the meniscus. The defect is then either treated withscaffold or scaffold+compound. The knee is straightened and the medialcollateral ligament is reattached with a screw and washer.

The knee is then flexed, and the mid-point of the lateral trochleasulcus is identified. The point of drilling for the cartilage defect isdefined as 20 mm distal to the proximal border of the lateral trochleargroove. The cartilage is scored with a 6 mm diameter punch, and usingspecialized instruments, a 6 mm diameter by approximate 2 mm deep defectis made in the cartilage surface. This defect is then either treatedwith scaffold or scaffold+compound.

With the knee still flexed, a collared 3 mm diameter bit is used todrill a pilot hole in the epicondylar region of the lateral femoralcondyle to a depth of 20 mm. The drill bit is aligned perpendicular tothe joint line and parallel to the anterior surface. This pilot hole isthen enlarged to a diameter of 8 mm. The bone defect is flushed and theneither treated with a scaffold or scaffold+compound.

Following closure of the surgical incision in 3 layers using 1-0 Vicrylfor the deep layers and skin staples, a modified Thomas splint isapplied to the leg to limit weight bearing and motion. The fiberglasscast and splint will remain on for a minimum of 14+2 dayspost-operatively. During this time animals will be maintained in smallpaddocks.

TABLE 10 Treatment assignment per defect Number of Group AnimalsTreatment 1 1 Bone Defect: Compound Cartilage Defect: Compound MeniscalDefect: Compound 1 2 Bone Defect: Compound Cartilage Defect: CompoundMeniscal Defect: Compound 1 3 Bone Defect: Compound Cartilage Defect:Compound Meniscal Defect: Compound 1 4 Bone Defect: Scaffold CartilageDefect: Scaffold Meniscal Defect: Scaffold 2 5 Bone Defect: ScaffoldCartilage Defect: Scaffold Meniscal Defect: Scaffold 2 6 Bone Defect:Scaffold Cartilage Defect: Scaffold Meniscal Defect: Scaffold

Animals are euthanized under stage III anesthesia with PotassiumChloride IV at days 84+2, postoperatively. Following euthanasia, thestifle joints are grossly evaluated, synovial fluid evaluated grosslyfor color and viscosity, and samples collected as described in Table 12.The joints will be opened, photographed and the surface of the chondralsites scored as indicated in Table 13. The articulating surfacesopposing the defect sites will be examined for any abnormal jointsurface. Gross evaluation will be performed on the control and operatedknee joints. Popliteal lymph nodes and the synovial membranes will beexamined for any inflammation.

TABLE 11 Gross Evaluation and Sample Collection Gross Photograph SampleSample Evaluation and Score collection Right Popliteal lymph node XRight Knee Joint X X(H) cartilage and meniscus Left Popliteal lymph nodeX Left Knee Joint X X cartilage and meniscus H = histology - Only rightfemur: bone defect, trochlear defect, medial meniscus

H=histology—Only right femur: bone defect, trochlear defect, medialmeniscus

The contralateral knee is examined for any abnormal joint surface. Grossmorphological evaluations of the right knee joints are made to determinethe chondral surface repair based on previous scoring criteria listed inTable 3. The right femora is cut to separate the cartilage defect fromthe bone void region and placed into appropriately labeled containersfilled with a 10-fold volume of 10 percent neutral buffered formalin.The medial meniscus is evaluated grossly, harvested and placed intoappropriately labeled containers filled with a 10-fold volume of 10percent neutral buffered formalin.

TABLE 12 Scoring Criteria for Gross Morphological EvaluationsCharacteristic Grading Score Edge Integration Full 2 (new tissuerelative to native cartilage) Partial 1 None 0 Smoothness of thecartilage surface Smooth 2 Intermediate 1 Rough 0 Cartilage surface,degree of filling Flush 2 Slight depression 1 Depressed/overgrown 0Color of cartilage, opacity or Opaque 2 translucency of the neocartilageTranslucent 1 Transparent 0

Synovial fluid is collected, evaluated for volume, viscosity (string),clarity and color. As appropriate, a semi-quantitative scoring of thegross synovial fluid evaluation is applied as outlined in Table 13.

TABLE 13 Description and Score for Synovial Fluid Score Color ClarityString 0 S = STRAW C = CLEAR N = NORMAL 1 P = PINK H = HAZY A = ABNORMAL2 Y = YELLOW/R = RED D = CLOUDY W = WATERY 3 B = BLOODY T = TURBID

Total synovial fluid score is a sum of the color, clarity and stringscores (0-8 points).

Example 7—Cell Migration Assays Using Processed Rat Microvascular Tissue

To demonstrate the effect of the processed microvascular tissuecomposition on cell migration, assays of human endothelial cellmigration (chemoattraction by processed microvascular tissuecompositions) and labeled processed microvascular tissue compositionmicrovesicle (MV) uptake by adipose-derived stromal vascular fraction(SVF) cells were developed and used as measures of the composition'sbiological activity.

The rationale for choosing these studies was that: (1) the processedmicrovascular tissue composition may induce increases in vascularrepair; thus, endothelial cell migration would be a valid metric; and(2) MV release and uptake is an important activity that would occur inmultiple cell types in a tissue repair model; thus, the SVF cellpopulation, which has cell types important for vascular repair was usedto test MV uptake.

General Study Design

To assay chemoattraction ability of the processed microvascular tissuecomposition for endothelial cells, samples of the composition wereplaced into the bottom chambers of transwell plates, and migration ofendothelial cells labeled with an orange-red fluorescent lipophilic dye(CM-DiI) was monitored over time, from 12 to 48 hr. The assay alsoincluded a chemically defined cryopreservation medium EZ-CPZ™ (IncellCorp., San Antonio, Tex.) at 100% and as a 50/50 mix of EZ-CPZ™ withM3D™ (Incell Corp., San Antonio, Tex.) media as a baseline control.

A second study was performed to assess uptake of the lyophilizedprocessed microvascular tissue composition by SVF cells. The compositionwas incubated with CM-DiI to allow the dye to be incorporated into theMVs and cell membranes of the composition samples. The labeledcomposition samples were rinsed, diluted in media, then placed ontoattached monolayer cultures of SVF cells. After 24 hr uptake the cellswere rinsed, then visualized for uptake of red fluorescent dye.

Materials and Methods

M3D™ is a chemically defined culture medium manufactured by INCELL. Insome assays M3D™ was supplemented with antibiotics (1×PSF:Pen/Strep/Fungizone antibiotic/antimycotic; Invitrogen, Grand IslandN.Y.). M3D:10 medium (INCELL) was M3D™ supplemented with 10% fetalbovine serum (FBS) and 1×PSF. EZ-CPZ™ (Incell Corp., San Antonio, Tex.)is a chemically defined cell cryopreservation medium. EZ-CPZ™ andEZ-CPZ™:M3D™ (1:1; v/v) were used as reference control media.

CM-DiI is the “Cell Tracker®” fluorescent dye in an aqueous formulationfor culture medium (Invitrogen/Molecular Probes). It becomes associatedwith lipophilic materials such as cell membranes and MVs and can bevisualized as bright red fluorescence by fluorescence microscopy. Forthis study, an EVOS inverted microscope was used with a red filter: 530nm excitation, 593 nm emission).

Human umbilical vein endothelial cells (HUVEC) at passage 1 (p1) wereretrieved from the INCELL Biorepository. Human SVF p1 cells for the MVuptake assays were retrieved from the INCELL Biorepository.

All cells were grown in M3D:10™ (Incell, San Antonio, Tex.). Endothelialcells were incubated with CM-DiI for 30 minutes at 37° C. and thenrinsed with M3D™ with 1×PSF. Cells were counted and an equal number ofcells per well was applied to the top chamber of 3 micron pore size PETtranswell chambers (Thermofisher; Waltham, Mass.) pre-loaded with theprocessed microvascular tissue composition test materials in triplicatewells.

The SVF cells for the adsorption assay were grown in 48-well plates intolog phase growth.

Processed Microvascular Tissue Composition

Two processed microvascular tissue compositions were prepared andtested, i.e., BMA and BMB. BMA samples were rat SVF cells, and BMB wererat bone marrow mononuclear cells each lyophilized at 10⁶ cells/ml. Therat SVF cells were prepared by mincing epididymal fat pads with scissorsuntil no pieces were larger than 1 mm in diameter then washed andincubated in 1 U/ml CIzyme AS (Vitacyte, Indianapolis, Ind.) in PBS for60 min at 37 C with gentle agitation. The cells were washed twice andresuspended at 10⁶/ml in lyophilization buffers. Rat bone marrow cellswere obtained by flushing the femurs and tibias with ACDA/PBS solutionand dissociated by repeated aspiration and expulsion through a 20 ganeedle. The bone marrow cells were then separated on a Ficoll gradient,washed with PBS+1% FBS twice and resuspended in buffers. Followinglyophilization, the BMA samples gave cell counts below the detectionlimit (under 10,000), while the BMB samples gave cell counts of 0.6 to1.0 million and viabilities (trypan blue) of 10 to 50%, as shown inTable 14.

TABLE 14 Viability of BMA and BMB Preparations # Cells Samples Pre-lyophPost-lyoph Viability (%) #Vials BMA Buffer 1 45,000 2,500 80 3 BMABuffer 2 27,000 7,500 0 3 BMA Buffer 1 4.4 × 10⁵  6 × 10⁵ 48 4 BMABuffer 2   5 × 10⁵ 10 × 10⁵ 10 4

All samples were stored refrigerated in the dark for a year, and testsamples were sterilized with 11 kGy E-beam radiation. Non-irradiatedcontrol samples were not sterilized.

Transwell Migration of Labeled Endothelial Cells

The lyophilized material was reconstituted in 1 ml of UFDI water with1×PSF. Of this total sample, 300 μl was placed in the bottom of each of3 wells and brought to a final volume of 500 μl with 200 μl M3D. Theupper chambers were set into the sample wells, and an equal number ofCM-DiI labeled HUVEC p2 cells was placed in each one. The plate wasincubated at 37° C., and wells were imaged at 12, 24 and 48 hours. Theimages were examined by counting cells in fields to determine theresults.

Uptake of Labeled Microvesicles by SVF Cells

The remaining 100 μl of lyophilized material was incubated with CM-DiIto label the cell membranes present. The material was washed 3 timeswith M3D+1×PSF by centrifugation. SVF cells seeded onto 48-well platesfor the absorption assay were grown to 50% confluence and had 50p1CM-DiI labeled lyophilized material layered on top. Half of the wellswere rinsed at 24 hours, coated with liquid mount and allowed to dry;the other half was rinsed, fixed and mounted after 3 days. The imageswere examined to determine the results.

Results

Transwell Migration of Labeled Endothelial Cells

For all of the wells, there were minimal numbers of cells in the lowerchamber at the 12 hour time point. See FIG. 2 and the first row of FIGS.3-7 as time progressed, the cells started to migrate into the lowerchamber. Some of the samples were drawn to the lower chamber morequickly. The BMA samples tended to attract cells more than the BMB asseen in FIG. 2. BMA Buffer 2 test group had the lowest cell number at 24hours but the highest cell counts at the 48 hour time point among thetest samples. Overall the BMA samples worked better than the BMB sampleswith buffer 2 having exhibiting a trend (but not statisticallysignificant) of induction of higher migration rate than buffer 1.

The other noticeable effect of the assay was the trend for a decrease inthe migration irradiation caused in BMA of about 20%, whereas BMB hadlittle effect and was essentially the same level (the difference was notstatistically significant) at 24 and 48 hours.

The EZ-CPZ™ media control did have some transmigration but only later inthe time course and to a lesser degree than the BMA and BMB samples.These results demonstrate dramatically that the cells do not have to beviable or autologous for the composition to induce an angiogenicactivity.

Uptake of Labeled Processed Microvascular Tissue CompositionMicrovesicles by SVF Cells

This assay was performed to determine if processed microvascular tissuecomposition MVs were transported into SVF (stromal vascular fraction)cells. The assay was performed by labeling the remaining 100 μl ofprocessed microvascular tissue composition material in each vial withCM-DiI, which incorporates into cell membranes even if the cellsthemselves are dead. The SVF cells were plated in a 48 well plate withM3D:10 media and allowed to adhere and grow until they wereapproximately 50% confluent. The 50 μl of the labeled material was addedto the wells and incubated for 6 hours. The wells were rinsed 3 timeswith M3D™ and fixed with wet-mount. Images showed that several of thecells did indeed take up the material as the dye was transferred to theSVF cells as seen in FIGS. 8 and 9.

Discussion

The BMA and BMB lyophilized material with or without irradiation actedas chemo-attractants to endothelial cells in this assay. A mild decreasein cell transmigration when the material was irradiated was noticed. Thedifference between buffers 1 or 2 was negligible.

The BMA and BMB material was taken up by living SVF cells when thematerial was placed on top of an already growing culture and incubatedfor 24 hours.

There are several important concepts illustrated with these experiments.Although the SVF samples showed huge cell losses and no viabilityfollowing lyophilization, they retained more biological activity thanthe bone marrow samples, which showed no cell losses and viability of 10to 50%. (The SVF cell loss may have been caused by excessive enzymeactivity during the digestion step.) Both cell preparations were stablefor over a year. Sterilization did not materially affect their abilityto attract endothelial cells.

TABLE 15 Growth Factors, Receptors, Hormones, Genes, TranscriptionFactors Associated with Bone GROWTH FACTORS AND RECEPTORS IdentityExample Source General Description TGF-β SUPERFAMILY TGF-β 1 Genentech,Chiron, soft and hard tissue wound healing, oncology, Collagen/Celltrix,hematopoiesis, latent form of TGF-beta binds mannose-6-P receptor, colonand pancreatic cancers TGF-β 2 Genzyme T.R., macular holes, ulcers,oncology), betaglycan Celltrix TGF-β 3 Oncogene (B-M- Oncology, skeletalSquibb) TGF-β 4 (chicken) TGF-β 5 TGF-βR 1-3 Type III receptor aidsbinding to Type II receptor BMP 2 Genetics Institute KO lethal,expressed in tooth pulp BMP 3 (osteogenin) Urist, Reddi Aka osteogenin,G.I., expressed in lung BMP 4 (2a) KO lethal, expressed in lung, tooth:overexpr. = bone thickening (neonates) BMP 5 expressed in lung, shortear mutant mouse has bone deformities BMP 6 (vgr-1) expressed in lung,KO delayed ossification of sternum (mild phenotype) BMP 7 (OP-1)Creative Biomolecules KO kidney, eye; expressed in tooth pulp BMP 8(OP-2) Creative tooth pulp Biomolecules BMP 9 (OP-3) CreativeBiomolecules BMP 10 BMP 11 BMP 12 (GDF-7) G.I. Promoted cartilage andtendon growth, P-T repair BMP 13 G.I. Promoted cartilage and tendongrowth (GDF-6, CDMP-2) BMP 14 BMP 15 G.I. BMPR IA Type IA upregulatesType IB, KO ectopic bone and cartilage (no pheno in another lab) BMPR IBActivating IB gives cartilage (even ectopic); KO no cartilage. BMP2RType II receptor binds to type IA (increase nodules) or IB (lessnodules) GDF 1 expressed in brain, tooth pulp GDF 2 GDF 3 (Vgr-2) GDF 4GDF 5 soft tissue, tooth pulp, bone and cartilage (MP52, CDMP-1) GDF 6tendon, ligament, cartilage, thicker and longer (BMP-13, CDMP-2) bones(Gene TX to increase production in neonates), tooth pulp GDF 7 (BMP-12)cartilage and tendon, tooth pulp GDF 8(myostatin) MetaMorphix KO allows2-3X muscle mass increase GDF 9 GDF 10 Membranous bone, adipose tissueDpp Fruit fly protein analagous to BMP 2 and OP-1, Induces bone,cartilage, and bone marrow in mammals 60A Fruit fly protein analagous toBMP 2 and OP-1, Induces bone, cartilage, and bone marrow in mammals MP52Differentiation of mesenchymal progenitors, soft tissues, tooth pulpunivin Vg1 Vgr-1 (BMP-6) Expressed in lung Vgr-2 (GDF3 nodal fugacinADMP dorsalin-1 PC-3 radar screw lefty GDNF Expressed in tooth pulpα-inhibin KO's develop gonadal then adrenal tumors inhibin βA Inhibin βBInhibin βC Inhibin βD MIS Mullerian Inhibiting Substance CDMP 1Increases chondrogenesis and osteogenesis in vitiro, subQ cartil. andbone CDMP 2 Increases chondrogenesis in vitro, subQ cartil. and bone invivo. OGP Osteogenic Trauma factor which induces bone. 14-mer, lastpeptide (I. Bab). 5 AA's are essential. TGF-α Soft tissue wound healing,similar to EGF PDGF AA Chiron, Creative Chiron Phase III clinical showed50% reduction Biomolecules in wound size at 28 days, in clinicals forperiodontal disease PDGF BB Zymogenetics cartilage repair PDGF AB CTGF(connective Binds to PDGF-BB receptor on 3T3 cells tissue growth factor)CEF 10 45% homology to CTGF EGF Closely related actions to TGF-α HB-EGFRelated to TGF-α, potent in wound healing Pleiotrophin Induced bone andcartilage in rat calv defect (HB-GAM) VEGF Vascular Endothelial GrowthFactor -- secreted by osteoblasts FGF-1 (aFGF) Receptor interactionswith heparin, polyanions, matrix molecules (heparan sulphate) areimportant in action and regulation of the FGFs FGF-2 (bFGF) Synergenclinical study on soft tissue healing (poor results) Mundy showed boneformation following systemic injection. Orquest licensed from SCIOS,hyal acid carrier Ossigel inject for fracture repair FGF 3-6 Oncogeneproducts FGF-7 (KGF) Mesenchymal stimulation of skin growth and healing(keratinocytes) FGF-8 FGF-9 FGF-18 (Zfgf5) Zymogenetics Shown to havechondrogenic effect in vivo FGFR 1-4 NGF Osteopontin Metra (Orquest)possible induction of osteoprogenitors Osteopoietin Growth Hormone, actsvia IGF-I IGF I Genentech, skeletal growth and protein metabolism, bone(somatomedin C) Cephalon formation when infused in rats IGF II MSAEmbryogenesis, Form most common in mammals > rats IGFBP 1 IGFBP 2 IGFBP3 Celltrix Increased bone mass local and systemic IGFBP 4 IGFBP 5Chiron, Baylink, BMGmbh IGFBP 6 LDGF TNF-α INFα INFβ Chiron, Betaseronmarketed for MS INFγ consensus Genentech in clinical trials CSF-1Required for osteoclast formation IL-4 Inhibits OB function IL-6Imclone, Sandoz, Made by and activates OB and O'clst, Serono (2Xgp130)receptor IL-8 IL-11 [2Xgp130] receptor LIF (Leukemia made by andactivates OB, [LIFR + gp130] Inhibitory Factor) Oncostatin-M activateOB, increase differentiation [OSMR + gp130 or LIFR + gp130]Cardiotrophin-1 increase nodule formation [LIFR + gp130] CNTF [LIFR +gp130] ACTIVIN βA Tooth pulp ACTIVIN βB Tooth pulp Noggin Regeneron, P&GBinds to BMP's and antagonizes actions BNP (brain Scios Increasedchondroprogenitors in vivo, natriuretic peptide) ROBO-1 GlaxoUpregulated when vertebrae stretched, 37 kd, estrogen raises, PTH lowersVEGF-1 Einhorn found expression during Ilzarov technique BSP (boneHealed calvaria defect when immobilized to sialoprotein) gelatinOsteopontin Induced bone formation in rat calv defect Endothelin 1Expressed by OB and chondro (and endothelial), both have receptors.Endothelin 3 induced bone and cartilage formation in rat calv HORMONESPTH PTHR PTHrP Signals from the perichondrium to receptors in the growthplate chondrocytes to prevent hypertrophic differentiation = negativefeedback for Ihh, Bcl-2. PTHrP(107- bone induction region. KO lethal allcartilage 111) ossified and fused, Overexpress causes massive cartilageonlages Vitamin D metabolites Vitamin DRs Calcitonin Estrogen Raisesprolif. and differn. of marrow cells, less adipogenesis RA (retinoicDifferentiation acid) RAR α, β, γ receptors in nucleus. α and γ involvedin cartilage formation T4 (thyroid) Speeds differentiation of growthplate GH (growth Delays differentiation of growth plate hormone) PGE 1Local infusion caused bone formation PGE 2 EB 2 PG receptor on marrowcells EB 4 PG receptor on bone progenitors MISCELLANEOUS PGP CTAP-IIIb-TG NAP (1-3) PF-4 MGSA GRO IP10 C9E3 CEF-4 MMP-2 GENES HoxA-1 HoxA-2Second branchial arch, craniofacial elements HoxA-3 HoxA-4 HoxA-4 HoxA-6HoxA-7 HoxA-9 Shoulder HoxA-10 Humerus HoxA-11 Radius-ulna HoxA-13phalanges HoxB 1 HoxB 2 HoxB 3 HoxB 4 HoxB 5 HoxB 6 HoxB 7 HoxB 8 HoxB 9Shoulder HoxB 13 Phalanges HoxC 4 HoxC 5 HoxC 6 HoxC 8 HoxC 9 shoulderHoxC 10 Humerus HoxC 11 Raidus-ulna HoxC 12 Metacarpals HoxC 13Phalanges HoxD 1 HoxD 3 HoxD 4 HoxD 8 HoxD 9 Shoulder HoxD 10 HumerusHoxD 11 Radius-ulna HoxD 12 Metacarpals HoxD 13 phalanges, Homeoboxgene, 4th sacral vertebra Shh (Sonic skeletal patterning, induces FGF-4,BMP-2&4, hedgehog) HoxD-13 Ihh (Indian similar activity and signaling toShh, regulates hedgehog) (prevents) hypertrophic differentiation, (−)feedback via PTHrP, induces BMP2, PTC, GLI, HOXD-11, HOXD-13, repressescollagen type X and BMP-6. Dhh (Desert spermatocyte survival hedgehog)Pax 1 KO slight phenotype Pax 2 Pax 3 Pax 4 Pax 5 Pax 6 Pax 7 Pax 8 Pax9 KO gave no teeth or thymus, cleft palate, and extra thumb WNT 5AProximal-distal outgrowth of limbs under control of apical ectodermalridge and FGF WNT 7A Dorsal-ventral patterning via LMX-1a LMX 1aDorsal-ventral patterning, activated by WNT-7a and repressed by En-1 En1 Dorsal-ventral patterning MSX 1 (Hox7) Homoebox gene involved ingrowth of intramembranous bone, reg'd by BMP-4. KO mild pheno unlessMSX-2 also KO'd (Hox8, 1) Transcription factor, homeobox gene, involvedin suture closure, reg'd by BMP-4. Bone site specific even in adults.Inhibits chick OB differn. EtOH blocks msx-2 expression in development.KO mild pheno unless MSX-1 also KO'd MSX 3 c-jun c-ras junb egr-1 c-srcGene required for OC's to resorb bone c-fos Gene required for OC's toform dHand eHand twist ID Permo-1 TRANSCRIPTION FACTORS *bHLH Family oftranscription factors triggering lineage commitment (e.g., MyoD) paraxisearliest marker of cells which will become somites, epithelial marker?scleraxis prefigures the skeleton after paraxis, but KO doesn't formsomites. Overexpression favors chondrocyte pheno CK-ERG Precedesformation of cartilage SOX9 Transcription factor, bowed long bones,related to SRY MAD-1 BMP-2 signal in C2C12 AP-1 TGF NF-1 TGF SP-1 TGFSP-2 SP-3 TGF TIEG TGF, estrogen

Example 8—In Vitro and In Vivo Effects of Microvascular Tissue

As discussed above, various stem cell preparations have shown beneficialeffects in animal and clinical studies for a variety of indications. Inmany instances, the survival of the administered stem cells isrelatively poor. The present studies were therefore designed to addresswhether, as discussed in relation to several embodiments above, viablestem cells are needed in order to achieve some (or all) of thetherapeutic benefits associated with stem cells. The present studyemployed, microvascular tissue, a rich source of stem and progenitorcells, that was processed according to the methods disclosed above.

In brief, microvascular tissue was isolated from human cadaveric adiposetissue by mincing the tissue and subsequently enzymatically digestingthe minced tissue. The digested tissue was then centrifuged to removefat, resulting in microvascular tissue. The microvascular tissue wasresuspended in a cryopreservation buffer (1:1 mix of M3:DC and EZ-CPZmedias, INCELL Corporation, San Antonio, Tex.), dispensed into vials,and lyophilized or both lyophilized and radiation sterilized.

The resulting processed microvascular tissue was assayed for cellcounts, viability, phenotype, CFU-F, and bioactivity in angiogenesis andorthopedic models.

Results

The cell counts and viability of freshly isolated, lyophilized andlyophilized/sterilized microvascular tissue is shown in Table 16. Thephenotype of each preparation is shown in Table 17.

TABLE 16 Preparation Lyophilized + Isolated Lyophilized Sterilized CellCount (DAPI/gram of 1.2 ± 0.2 0.9 ± 0.1 1.0 ± 0.3 fat) million millionmillion Viability (Trypan Blue) 85 ± 5% 15 ± 1% 2 ± 1%

TABLE 17 Phenotype Type IV Collagen CD31+ CD34+ CD44+ CD45+ Fresh 65 ±5%  52 ± 7%  58 ± 4% 55 ± 10% 5 ± 1% Microvascu- lar Tissue Lyoph- 85 ±13% 73 ± 17% 65 ± 9% 61 ± 13% 11 ± 3%  ilized + Sterilized

The cell count/cell viability data clearly demonstrate that there is nota significant change in cell number based on the processing of themicrovascular tissue (e.g., whether fresh, dried, or dried andsterilized, the cell counts are roughly equivalent, based on nuclearuptake of DAPI DNA stain). However, lyophilization of the microvasculartissue significantly reduces the ability of the cells in themicrovascular tissue to exclude trypan blue. Lyophilization inducesapproximately a 70% reduction in the percentage of viable cells.Exposure to radiation further reduced the viability, such that onlyapproximately 2% of the cells in the microvascular tissue were viable.Further, when assayed for functional mesenchymal stem cells (using anestablished colony-forming unit-fibroblast (CFU-F) assay), no functionalmesenchymal stem cells were detected in the lyophilized or thelyophilized/sterilized preparations.

Interestingly, despite the unchanged cell number and the reduction inviability, the various processing methods altered the phenotype of thecells. As shown in Table 17, Type IV collagen increased modestly (versusa fresh preparation) in lyophilized/sterilized microvascular tissue.This data suggests that the lyophilized/sterilized microvascular tissuemay be well-suited for repair of soft tissues, due at least in part toits increased collagen density. Collagen-based materials have beentested for their use in tissue-engineering, however the microvasculartissue disclosed herein is particularly advantageous because of theready availability of the material, and its enhanced “stemness”(discussed below). Each of the established hematopoietic stem cellmarkers CD31 (hematopoietic stem cells), CD34 (bone marrow/hematopoieticstem cells), CD44 (cancer stem-like cells) and CD45 (hematopoietic stemcells) were essentially were unregulated in response to lyophilizationand sterilization. Thus, despite a nearly complete reduction in theviability of the cells in lyophilized/sterilized microvascular tissue,any cells remaining have enhanced expression of markers known to beexpressed by stem cells. As such, the lyophilized/sterilizedmicrovascular tissue may be more suited to tissue regeneration becauseof this enhanced stemness (and the indirect effects that the increasedstemness induces, e.g., paracrine recruitment of other endogenous cellsthat enhance repair, release of growth factors from the microvasculartissue, etc.).

The various microvascular tissues were evaluated for their ability torecruit other types of cells. Recruitment of cells can enhance therepair or regeneration of damaged or diseased tissue by a variety ofmechanisms. For example, recruitment of endothelial cells can enhanceblood vessel formation, thereby improving blood supply that facilitatesnew tissue formation. Recruitment of endogenous stem cells can initiatea cascade of events that foster new tissue growth and/or repair ofexisting damaged tissue. Human umbilical vein endothelial cells (HUVEC)labeled with DiI and were placed in the top of Transwell plates withvarious types of microvascular tissue in the bottom wells. The number ofHUVEC's crossing the intra-well membrane was counted for each type ofmicrovascular tissue after 48 hours and compared to culture media aloneor media supplemented with epidermal growth factor (EGF) as controls.FIG. 10 depicts the results. Little migration of HUVEC cells wasdetected in response to media alone. EGF, which is established as aninducer of HUVEC migration result in about 10 times more migration thatmedia alone. Freshly isolated microvascular tissue (“digested” in FIG.10) induced slightly less migration than EGF, and lyophilized inducedeven less migration (though it was still greater than media alone).Unexpectedly, lyophilized/sterilized microvascular tissue induced nearly2 times more migration as compared to EGF, and nearly 20 times more thanmedia alone. Thus, the drying and sterilization of microvascular tissuesignificantly enhances its ability to recruit cells in vitro. Thatenhanced ability, in several embodiments, provides, at least in part,enhanced tissue repair and/or regeneration in vivo.

That enhanced repair in vivo was corroborated by demonstrating thatlyophilized/sterilized microvascular tissue induces a more robust andrapid restoration of blood flow to tissue that was rendered ischemic.SCID mice were subjected to unilateral ligation and transection of thefemoral artery according to established methods in order to replicateischemia (a condition that leads to severe tissue damage). Microvasculartissue processed in various ways was introduced into the ischemic limbon day 0, 3, and 7, and the mice were imaged by laser doppler on days 0,7, and 14. As shown in FIG. 11, control animals injected with salineshow little restoration of blood flow (the ischemic limb is designatedwith an arrow) after 7 or 14 days. In contrast, lyophilizedmicrovascular tissue resulted in at least partial restoration of bloodflow by 14 days. However, lyophilized/sterilized microvascular tissueresulted in significant increases in blood flow by 7 days, with bloodflow at 14 days largely indistinguishable from the contralateral controllimb. These data corroborate the in vitro migration data and indicatethat, in several embodiments, the lyophilized/sterilized microvasculartissue can enhance restoration of blood flow.

In several embodiments, the restoration of blood flow is due, at leastin part, to formation of new blood vessels, including small vessels(e.g., microvasculature), medium vessels, and large diameter vessels(e.g., those that are major suppliers of blood to a tissue). Matrigel(0.5 mL) was mixed with saline or microvascular tissue (human) togenerate a microvascular tissue implant, which was injectedsubcutaneously into SCID mice. After 14 days the implants were removed,fixed, and stained with a-CD31 fluorescent antibody. Blood vessels thathad infiltrated the implants were sized and counted with a microscope.Standard deviations were 12-30% of average counts/field. 2 doses oftissue were tested after each processing step. There were no human CD31+cells found.

FIG. 12 summarizes the data related to infiltration of various vesselsizes after implantation of microvascular tissue implants having variouscell numbers and being processed in different manners. Matrigel resultedin about 35 vessels per field, the majority being small vessels.Implantation of lyophilized microvascular tissue with about 50,000 cellsyielded increased infiltration, with a more robust generation of mediumsized vessels. Implantation of lyophilized microvascular tissue withabout 500,000 cells led to still further vessel generation, with asubstantial increase in the number of large diameter vessels.Implantation of lyophilized/sterilized microvascular tissue with about50,000 cells resulted in increased vessel numbers as compared tocontrol, with some generation of large vessels. Implantation oflyophilized/sterilized microvascular tissue with about 500,000 cellsresulted in significant vessel generation, over two times greater thancontrol. Interestingly, as compared to the 50,000 cell dose, the largercell number, despite the near-zero viability, yielded a number of largediameter vessels. Thus, administration of processed microvasculartissue, whether lyophilized or lyophilized/sterilized, appears to boostthe formation of larger diameter vessels. These data are furthersupportive of the ability of microvascular tissue to enhance themigration and/or formation of blood vessels, which is an importantaspect of repairing tissue. Moreover, in several embodiments, thegeneration of blood vessels of various sizes ensures that not only isthere adequate capacity of carry blood from the main branches of thecirculatory system to the target tissue (large diameter), but that bloodcan be effectively distributed throughout the target tissue, even tointerior portions (medium and small vessels).

Taken together, these data indicate that microvascular tissue has thecapacity to induce angiogenesis both in vitro and in vivo. As such,microvascular tissue is a highly attractive mechanism by which toinstitute tissue repair and/or regeneration, based on its ability toenhance angiogenesis, which will facilitate and/or maintain the repairand/or regeneration of tissue by ensuring adequate blood supply andnutrient/oxygen flow.

Additionally, microvascular tissue was assessed for its ability toenhance repair of bone and cartilage. With respect to bone, 8 mm×2 cmcritical-sized defects were drilled into the distal metaphysis of maturegoats. Tissue scaffolds (BIOFIBER, Tornier) were rolled tightly andinserted into the defects alone, or including microvascular tissue(volume of 1 ml, ˜10⁶ cells). Cells were loaded simply by adding 1 mlwater to vial, swirling briefly, then dripping the contents onto ascaffold and waiting 5 min for binding. At 12 weeks the defects werecompression tested and decalcified for histology.

FIGS. 13A-13C show data related to the repair of bone defects. FIG. 13Ashows the strength, elastic modulus, and toughness of the repaired boneas compared to the contralateral control. Use of the scaffold aloneresulted in the damaged bone having approximately 20% of the strengthand elastic modulus of the control bone, and roughly 50% of thetoughness of the control bone. When the scaffold was supplemented with alyophilized/sterilized microvascular tissue, each of strength, elasticmodulus, and toughness was increased relative to scaffold alone. Thesedata thus indicate, the use of microvascular tissue facilitates therepair of damage to bone. FIGS. 13B and 13C, show representativehistology from bones treated with the scaffold alone and bones treatedwith the scaffold supplemented with microvascular tissue. FIG. 13B(scaffold alone) shows some initial osteophyte formation at the junctionbetween the bone and the fiber scaffold that was placed within thedefect. This suggests that the fiber scaffold institutes at least somebone repair, as evidenced by the data of FIG. 13A. In FIG. 13C, thehistology suggests that the addition of the lyophilized/sterilizedmicrovascular tissue resulted in true bone formation at the margins ofthe scaffold. Thus, within the same time period, microvascular tissuefacilitated the formation of bone, rather than the demineralizedprecursor to bone. This suggests that the use of microvascular tissue inconjunction with a scaffold can accelerate the healing process.

With respect to cartilage repair, 4 mm×7 mm critical-sized defects werepunched into the cartilage on the medial trochlear groove of maturegoats. The defects were approximately 1 mm deep, which is slightlydeeper than the cartilage. Tissue scaffolds (BIOFIBER, Tornier) eitheralone or supplemented with lyophilized/sterilized microvascular tissue(˜10⁶ cells) were fitted into the defects and held in place with a 7-0nylon suture at each corner. After 3 months the defects were examinedhistologically. This data is shown in FIGS. 14A-14H. FIGS. 14A-14D showdata from the scaffold alone, while FIGS. 14E-14H show data from themicrovascular tissue supplemented scaffold. FIG. 14A shows a macroscopicview of the scaffold on the previously damaged cartilage, while FIG. 14Eshows the same view for the scaffold supplemented with microvasculartissue. Both treatment groups showed evidence of repair. FIGS. 14B and14F show hematoxylin and eosin staining of the cartilage. The use of thescaffold including microvascular tissue showed improved fill in marginsas compared to the use of scaffold alone. FIGS. 14C and 14G showsafranin O staining, and reveal a greater degree of proteoglycans andretention in the defects treated with microvascular tissue. FIGS. 14Dand 14H show toluidine blue staining of the defects, and reveal that thenewly generated cartilage matrix stains more like mature cartilage whenmicrovascular tissue was used to treat the defect. Together these data,as with the bone experiments above, confirmed that microvascular tissuefacilitates the repair of cartilage.

Experiments were also performed to evaluate the ability of microvasculartissue to repair tendon. Rat achilles tendons were exposed and abradedwith mouse-tooth forceps. Controls were operated on in the same manner.One group of rats was treated with scaffold alone and another withscaffold supplemented with lyophilized/sterilized microvascular tissue.Rats were sacrificed for qPCR, histology and immunohistochemistry after7 days. The Scaffold group received a 4 mm×7 mm BIOFIBER-CM Scaffold onthe anterior surface of the Achilles. The scaffold was loaded with 106microvascular cells in the microvascular tissue and treatment group.Control data is shown in FIGS. 15A (Masson's trichrome stain) and 15B(immunohistochemistry for tenascin). Data for the scaffold group isshown in FIGS. 15C and 15D. Masson's trichrome staining (FIG. 15C)revealed small pockets of dense collagen in and around the scaffold,indicative of initial tendon repair. Similarly, immunohistochemistrystaining for tenascin (FIG. 15D) revealed increases in expression at themargin between the tendon and implanted scaffold, again suggestive ofinitial repair of the defect.

Data for the microvascular tissue group is shown in FIGS. 15E and 15F.Masson's trichrome staining (15E) revealed substantial formation ofdense collagen in and around the scaffold, indicative of considerablerepair of the defect. Similarly, immunohistochemistry staining fortenascin revealed extensive expression between the tendon and implantedscaffold. These data are also indicative of significant repair of thedefect.

Example 9: Characterization of a Multipotent Cell Preparation

Multipotent cell products can be derived from many tissues and evendifferent species. They can be freshly-isolated or expanded in culture.They can even be generated by genetic manipulation of somatic cells (egiPSC). Because of the modest effects of PRP and BMAC vs MSC and SVF wetargeted a large dose of multipotent cells. There is no single,convenient marker for stem cells. The commonly used antibodies targetmarkers (such as CD34, CD90, CD117, CD271) that are found on stem cellsbut also on other cell populations. Thus, a cocktail of antibodies isoften used and cells that bind all of them (and do not bind a few morethat are never found on stem cells) are defined as stem cells. Thisapproach often overestimates the number of stem cells. A traditionalmethod for quantifying stem cells requires plating cell mixtures in alimiting dilution assay and then counting the number of fibroblast-likecolonies (CFU-F) that eventually grow. This method underestimates thenumber of stem cells because some may not grow in the cultureconditions. Acknowledging these concerns we targeted a dose of at least1000 CFU-F or 10⁵ CD90⁺ cells per square cm of wound.

When multipotent cell products are used to treat DFU, they are typicallyinjected into the wound bed and also around the wound periphery.Multipotent cells (PRP or BMAC) may also be applied topically to woundswith fibrin glue to hold them at the site. Such treatments have beenreported to improve wound healing, but have shown no effect onneuropathy.

When multipotent cells are used to treat neuropathy, they are usuallyinjected into the tissue adjacent to the damaged nerve or givensystemically. On some occasions when nerve damage is treated surgically,multipotent cells have been included in the nerve guide tubes used tohelp repair the nerve.⁸ There are some reports of success with suchtreatments, but not in diabetic neuropathy patients.

Herein, we applied large doses of multipotent cells topically to DFU andobserved a distinct improvement in healing of the DFU and surprisingly adramatic reversal of the peripheral neuropathy in the treated limb.

We have found that multipotent cells can be freeze-dried and sterilizedwhile maintaining or even enhancing their therapeutic benefits. (SeeU.S. application Ser. No. 15/633,311 filed Jun. 26, 2017; U.S.application Ser. No. 14/429,511 filed Mar. 19, 2015; PCT/US2013/060181filed Sep. 17, 2013). When processed in this manner the multipotentcells have important advantages over live freshly isolated or culturedcells: 1) stable for years when stored at room temperature, 2) ready touse in a minute or less, 3) effective in allogeneic or even xenogeneicrecipients, 4) no risk of disease transmission, and 5) no danger ofinappropriate differentiation or expansion of the cells.

When we applied these processed multipotent cells to diabetic footulcers (DFU), we again saw a dramatic improvement in wound healing and areversal of the peripheral neuropathy in that treated limb. The greatestimprovement in healing and in neuropathy was seen when the processedmultipotent cells were applied topically directly to the DFU, but therewas also improvement when they were injected locally.

The aim of these experiments was to develop and test convenient newproducts for treating DFU based upon multipotent cells. Acknowledgingconcerns regarding low cell number, we targeted a dose of at least 1000CFU-F or 10⁵ CD90⁺ cells per square cm.

A batch of multipotent cells was produced and vialed and thencharacterized for phenotype and some growth factors of interest atcontract labs. Cell count and phenotype were measured by INCELL, Inc.(San Antonio, Tex.) while growth factors were measured by ELISA atAssayGate (Ijamsville, Md.). The phenotype results show 5.5×10⁵ CD90⁺cells/vial. The cells were lysed and assayed by ELISA in a total volumeof 1 ml giving the results tabulated below. The HDGF (hepatoma derivedgrowth factor) was discovered at the Proteomics Lab at Colorado StateUniv.

TABLE 18 Growth Factor Quantity/vial bFGF 558 pg BDNF 40 pg TGF-β₁ <6.5pg HDGF 24 ng

Example 10 Topical Delivery to a Wound in a Limb with Neuropathy

The preparation of multipotent cells as characterized in Example 9 waslyophilized and sterilized by gamma irradiation. The resulting cake wasporous, brittle and easily crushed to powder. The powder was sprinkledon diabetic foot ulcers after debridement and cleaning per standardwound care practice. The wounds were then covered with an occlusivedressing (Adaptic Touch®, KCI, San Antonio, Tex.) held in place withSteri-Strips® (3M, St. Paul, Minn.). Two patients received themultipotent cell preparation while a control patient received the samecare without the addition of the multipotent cells. Healing of the DFUwas measured weekly before reapplication of the multipotent cells anddressings. The extent of the patients' peripheral neuropathy wasmeasured using the Von Frey method. A monofilament fiber that bends at acalibrated force is used to probe the limb, moving up the leg until thepatient can feel the fiber.

FIG. 16 shows results for Patient A who presented with a diabetic footulcer on the bottom of his foot that had not healed despite standardcare with hydrocolloid dressings. After debridement, the ulcer measured5.1 cm² and the neuropathy extended halfway to the knee. Four weekslater, the DFU measured 3.5 cm² and the extent of the neuropathy wasreduced by about 14 cm. In this patient, the reversal of neuropathy wasaccompanied by rather severe pain requiring medication. Weeklyapplication of the therapeutic agent was stopped when the DFU fullyhealed, but the neuropathy continued to heal and the associated painfully resolved subsequently.

FIG. 17 shows results for Patient B who presented with a diabetic footulcer on the medial side of her foot that had not healed despitestandard care with hydrocolloid dressings. After debridement the ulcermeasured 6.1 cm² and the neuropathy extended over halfway to the knee.Four weeks later the DFU measured 0.4 cm² and the extent of theneuropathy was reduced by about 16 cm. Her wound did fully heal and theimprovement in neuropathy persisted after completion of the treatmentswith the multipotent cell preparation.

FIG. 18 shows control data in which Patient C presented with a diabeticfoot ulcer on the bottom of her foot that had not healed despitestandard care with hydrocolloid dressings. After debridement the ulcermeasured 4.0 cm² and the neuropathy extended ⅓ of the distance to theknee. Four weeks later, the DFU measured 1.6 cm² and the extent of theneuropathy was reduced by about 1 cm. At 12 weeks, the DFU had nothealed and there was little change in her neuropathy.

As illustrated in FIGS. 16 and 17, the multipotent cell treatments had adramatic impact on the patients' neuropathy. By week 5, both patients Aand B had some feeling on the bottom of their feet for the first time inyears. Control patient C saw little effect.

Example 11: Subcutaneous Injection in Limb with Neuropathy

An adult male with diabetic neuropathy had lost much of his feelingbelow his left ankle. He was treated with 4 subcutaneous injections ofthe same preparation that was used in Example 10. Each injectionconsisted of 250,000 CD90⁺ cells dissolved in 1 ml of normal saline. Thepatient later reported a 60% improvement in sensation in his left footand the improvement extended from his ankle to the region of theinjections. Thus, the preparation did have a beneficial impact on theneuropathy, but it was not as pronounced as when the agent was appliedtopically to a DFU.

Taken together, the data presented in these experiments establish thatmicrovascular tissue is capable not only of angiogenesis (which plays animportant role in establishing and maintaining blood supply to a targettissue), but are also capable of enhancing the repair of bone,cartilage, tendon, and nerve. In several embodiments, the enhancedangiogenesis, at least in part, plays a role in the ability ofmicrovascular tissue to result in the generation and maintenance of newtissue.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety to the extent notinconsistent with the present description.

It is contemplated that various combinations or subcombinations of thespecific features and aspects of the embodiments disclosed above may bemade and still fall within one or more of the inventions. Further, thedisclosure herein of any particular feature, aspect, method, property,characteristic, quality, attribute, element, or the like in connectionwith an embodiment can be used in all other embodiments set forthherein. Accordingly, it should be understood that various features andaspects of the disclosed embodiments can be combined with or substitutedfor one another in order to form varying modes of the disclosedinventions. Thus, it is intended that the scope of the presentinventions herein disclosed should not be limited by the particulardisclosed embodiments described above. Moreover, while the invention issusceptible to various modifications, and alternative forms, specificexamples thereof have been shown in the drawings and are hereindescribed in detail. It should be understood, however, that theinvention is not to be limited to the particular forms or methodsdisclosed, but to the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the various embodiments described and the appended claims.Any methods disclosed herein need not be performed in the order recited.The methods disclosed herein include certain actions taken by apractitioner; however, they can also include any third-party instructionof those actions, either expressly or by implication. For example,actions such as “administering microvascular tissue” include“instructing the administration of microvascular tissue.” The rangesdisclosed herein also encompass any and all overlap, sub-ranges, andcombinations thereof. Language such as “up to,” “at least,” “greaterthan,” “less than,” “between,” and the like includes the number recited.Numbers preceded by a term such as “about” or “approximately” includethe recited numbers. For example, “about 3 mm” includes “3 mm.”

What is claimed is:
 1. A method for the treatment of damaged nervescomprising delivery of a therapeutic agent to a subject in need thereofwherein the therapeutic agent comprises enriched, nonviable multipotentcells.
 2. The method of claim 1 wherein the multipotent cells aresterilized by irradiation.
 3. The method of claim 1 wherein themultipotent cells have been isolated from microvascular tissue.
 4. Themethod of claim 1 wherein the multipotent cells have been enriched by amethod selected from the group consisting of removal of extraneoustissue and culturing the multipotent cells.
 5. The method of claim 1wherein the multipotent cells have been enriched by culturing themultipotent cells.
 6. The method of claim 1 wherein delivery of atherapeutic agent comprises injection or surgical application to theregion of damaged nerves.
 7. The method of claim 1 wherein delivery of atherapeutic agent comprises systemic injection.
 8. The method of claim 1wherein the therapeutic agent is a powder, gel or ointment appliedtopically near the site of nerve damage.
 9. A wound healing compositioncomprising sterilized microvascular tissue formulated for topicaladministration, wherein the microvascular tissue comprises multipotentcells, has angiogenic or anti-inflammatory activity greater than salinealone, and wherein less than or equal to 50% of the multipotent cellsare viable.
 10. The composition of claim 9 wherein the microvasculartissue is sterilized by irradiation.
 11. The composition of claim 9wherein the microvascular tissue comprises enriched multipotent cells.12. The composition of claim 9 wherein the microvascular tissue isenriched by a method selected from the group consisting of removal ofextraneous tissue or culturing the multipotent cells.
 13. A method oftreating an ischemic wound in a subject in need thereof, comprising:identifying a subject with an ischemic wound; and administering atherapeutically effective amount of a composition comprising sterilizedmicrovascular tissue formulated for topical administration, wherein themicrovascular tissue comprises multipotent cells, has angiogenic,anti-inflammatory, and/or tissue healing activity greater than salinealone, and wherein less than or equal to 50% of the multipotent cellsare viable.
 14. The method of claim 13 wherein the topical compositionis applied at least twice in a three month period.
 15. The method ofclaim 13 wherein the topical composition is applied at intervals of 3 to15 days.
 16. The method of claim 13, wherein the wound is selected fromthe group consisting of a surgical wound, a burn, an injury, a graftwound, a diabetic wound and a diabetic ulcer.
 17. The method of claim 13wherein said subject has a skin lesion.
 18. The method of claim 17,wherein said skin lesion is a venous ulcer, diabetic ulcer, pressuresore, burn or iatrogenic grafting wound.
 19. The method of claim 13,wherein the sterilized microvascular tissue comprising multipotent cellsis injected into a subcutaneous or dermal site.
 20. The method of claim13, wherein the sterilized microvascular tissue comprising multipotentcells is provided on a scaffold or bandage.